Flow through electroporation modules and instrumentation

ABSTRACT

The present disclosure provides a flow-through electroporation device configured for use in an automated multi-module cell processing environment and configured to decrease cell processing time and the risk of clogging.

RELATED CASES

The present application claims priority to U.S. Ser. No. 62/864,368,filed 20 Jun. 2019, entitled “Flow Through Electroporation Modules andInstrumentation”; and U.S. Ser. No. 62/964,203, filed 22 Jan. 2020,entitled “Flow Through Electroporation Modules and Instrumentation”,both of which are incorporated herein in their entirety.

FIELD OF THE INVENTION

The present disclosure relates to flow-through electroporation devicesconfigured as stand-alone electroporation modules or as one module inautomated multi-module cell processing instruments.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that thearticles and methods referenced herein do not constitute prior art underthe applicable statutory provisions.

The cell membrane constitutes the primary barrier for the transport ofmolecules and ions between the interior and the exterior of a cell.Electroporation, also known as electropermeabilization, substantiallyincreases cell membrane permeability in the presence of a pulsedelectric field. Traditional electroporation systems have been widelyused; however, traditional systems require high current input and sufferfrom adverse environmental conditions such as electric field distortion,local pH variation, metal ion dissolution and excess heat generation,all of which may contribute to low electroporation efficiency and/orcell viability. Further, traditional electroporation systems are noteasily automated or incorporated into automated cell processing systemswhere electroporation is but one process of many processes performed.There is thus a need for automated multi-module cell processing systemsand components thereof capable of transforming multiple cells in anefficient and automated fashion. The present invention addresses thisneed.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key or essentialfeatures of the claimed subject matter, nor is it intended to be used tolimit the scope of the claimed subject matter. Other features, details,utilities, and advantages of the claimed subject matter will be apparentfrom the following written Detailed Description including those aspectsillustrated in the accompanying drawings and defined in the appendedclaims.

The present disclosure provides an electroporation device configuredboth for use as a stand-alone electroporation device and for use in anautomated multi-module cell processing environment. The electroporationdevice utilizes a microfluidic flow-through configuration thatfacilitates continuous processing of cell suspensions. By decreasing thecross-sectional area of paths or lanes of fluid flow in the flow channelbetween electrodes and adjusting the pressure driving the fluid flowthrough the device, the electric field strength experienced by cells canbe made sufficiently high with a desired duration of poration to poratethe plasma membrane. The narrow flow channels are parallelized todecrease processing time and reduce the chance of catastrophic failuredue to clogging.

In certain embodiments, there is provided a flow-through electroporation(FTEP) device for introducing an exogenous material into cells in afluid, the FTEP device comprising: an inlet and an inlet channel forreceiving a fluid comprising cells and/or exogenous material into theFTEP device; an outlet and an outlet channel for removing a fluidcomprising transformed cells and exogenous material from the FTEPdevice; a flow channel intersecting and positioned between the inletchannel and the outlet channel, wherein the flow channel has, movingfrom the inlet channel toward the outlet channel, an inlet-filterregion, an inlet-proximal region, a central region, an outlet-proximalregion, and an outlet-filter region; an inlet filter comprising filterelements disposed in the inlet-filter region of the flow channel and anoutlet filter comprising filter elements disposed in the outlet-filterregion of the flow channel; a plurality of obstructions defining flowpaths disposed within the central region of the flow channel; and afirst and a second electrode positioned in electrode channels, whereinthe first electrode is positioned in the inlet proximal region of theflow channel and the second electrode is positioned in the outletproximal region of the flow channel; wherein the electrodes arepositioned perpendicularly to the flow channel, are in fluid andelectrical communication with fluid in the flow channel, and wherein theelectrodes apply one or more electric pulses to the cells in the fluidas they pass through the flow channel, thereby introducing exogenousmaterial into the cells in the fluid.

In some aspects of this embodiment, the plurality of obstructionsdefining flow paths disposed within the central region of the flowchannel increase in density from the inlet proximal region of the flowchannel to a central portion of the central region and decrease indensity from the central portion of the central region to the outletproximal region of the flow channel.

In some aspects of this embodiment, the FTEP device further comprises areservoir coupled to the inlet for introducing the cells in fluid intothe FTEP device and a reservoir coupled to the outlet for removingtransformed cells from the FTEP device, and in some aspects, the FTEPdevice comprises a second inlet and a second inlet channel and furthercomprises a reservoir coupled to the second inlet for introducingexogenous material into the FTEP device. In some configurations, thesecond inlet and second inlet channel are located between the inletchannel and the first electrode, and in alternative configurations thesecond inlet and second inlet channel are located between the firstelectrode and the central region.

In some aspects, elongated obstructions may be arranged in a parallelconfiguration, and in some aspects, peg-like obstructions may bearranged in rows.

The FTEP devices described herein are configured for use with bacterial,yeast and mammalian cells.

In some aspects, the number of obstructions in the central region of theflow channel is from 5 to 100, and in other aspects, the number ofobstructions in the central region of the flow channel is from 6 to 80,7 to 60 or 8 to 40.

In some aspects, the inlet filter region comprises an inlet filtercomprising filter elements disposed in the inlet-filter region of theflow channel, wherein the inlet filter elements comprise triangular-,square-, rectangular-, pentagonal-, hexagonal-, oval-, orelliptical-shaped elements.

In some aspects, the outlet filter region comprises an outlet filtercomprising filter elements disposed in the outlet-filter region of theflow channel, wherein the outlet filter elements comprise elongatedoval-, triangular-, square-, rectangular-, pentagonal-, hexagonal-,oval-, or elliptical-shaped elements.

In some aspects, the narrowest flow path between obstructions is from 10μm to 350 μm wide, and in other aspects, narrowest flow path betweenobstructions is from 30 μm to 250 μm wide.

In some aspects, the obstructions are triangular-, square-,rectangular-, pentagonal-, hexagonal-, oval-, or elliptical-shapedobstructions, or a combination of two or more shaped obstructions.

In many aspects, there is presented herein an automated multi-modulecell processing instrument comprising the FTEP devices described herein.

In some aspects, the FTEP device demonstrates optimal uptake at 6.8 psiand 3 kV and recapitulates a rectangular pulse and residence timesimilar to a cuvette system.

In some aspects of the FTEP device, the electrodes supply a voltage of1-25 kV/cm, and in some aspects, the electrodes supply a voltage of 5-60kV/cm, or 10-50 kV/cm, or 20-40 kV/cm.

In some aspects, the flow through the FTEP device is from 0.01 mL/min to7.5 mL/min and the pressure in the FTEP is from 1-30 psi, or from 2-10psi. In some aspects, the FTEP is from 3-15 cm long, or from 4-12 cmlong, and from 0.5 to 5 cm wide.

In some aspects, the FTEP device further comprises a ramp in the centralregion proximal to the inlet-proximal region of the flow channelextending to a central portion of the central region, wherein the flowchannel height decreases at the central portion, and a ramp from thecentral portion of the central region to the central region proximal tothe outlet-proximal region of the flow channel wherein the flow channelheight increases at the outlet-proximal region. In some aspects, theFTEP device further comprises steps instead of ramps wherein thedecrease in channel height from the inlet-proximal region to the centralregion and the increase in channel height from the central region to theoutlet-proximal region is an abrupt step rather than a ramp.

These aspects and other features and advantages of the invention aredescribed below in more detail.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the presentdisclosure will be more fully understood from the following detaileddescription of illustrative embodiments taken in conjunction with theaccompanying drawings in which:

FIGS. 1A through 1C are top perspective, bottom perspective, and bottomviews, respectively, of a flow-through electroporation device assembly.FIG. 1D depicts one embodiment of a bottom view of an FTEP, and FIG. 1Eis a blow up of the circled region of FIG. 1D. FIG. 1F-1H arerepresentations of an obstruction array and individual obstructionstructures. FIGS. 11-1K are representations of an obstruction array andindividual obstruction structures. FIG. 1L-1N are representations of analternative embodiment of an obstruction array and individualobstruction structures. FIG. 1O depicts (i) electrodes arrayed on anelectrode platform before insertion into an FTEP assembly; (ii) anelectrode; and (iii) the electrode inserted into an electrode channelwith the electrode and electrode channel adjacent to the flow channel.FIG. 1P shows scanning electromicrographs of an inlet-filter region of aflow channel comprising a filter, as well as an inlet.

FIGS. 2A and 2B depict the structure and components of an exemplaryembodiment of a reagent cartridge comprising an FTEP device.

FIGS. 3A-3F depict the structure and components of an exemplary FTEPdevice that is configured to reside within, e.g., the exemplary reagentcartridge shown in FIG. 2A.

FIGS. 4A-4C depict an automated multi-module instrument and modules andcomponents thereof with which to generate the edited cells.

FIG. 5A depicts one embodiment of a rotating growth vial for use with acell growth module. FIG. 5B illustrates a perspective view of oneembodiment of a rotating growth vial in a cell growth module. FIG. 5Cdepicts a cut-away view of the cell growth module from FIG. 5B. FIG. 5Dillustrates the cell growth module of FIG. 5B coupled to LED, detector,and temperature regulating components.

FIG. 6A depicts retentate (top) and permeate (bottom) members for use ina tangential flow filtration module (e.g., cell growth and/orconcentration module), as well as the retentate and permeate membersassembled into a tangential flow assembly (bottom). FIG. 6B depicts twoside perspective views of a reservoir assembly of a tangential flowfiltration module. FIGS. 6C-6E depict an exemplary top, with fluidic andpneumatic ports and gasket suitable for the reservoir assemblies shownin FIG. 6B.

FIG. 7A depicts a simplified graphic of a workflow for singulating,editing and normalizing cells. FIGS. 7B-7D depict an embodiment of asolid wall isolation incubation and normalization (SWIIN) module. FIG.7E depicts the embodiment of the SWIIN module in FIGS. 7B-7D furthercomprising a heater and a heated cover.

FIG. 8 is a flow chart of an exemplary method for automated multi-modulecell editing to produce the cell libraries as described herein.

FIG. 9 is a simplified block diagram of an embodiment of an exemplaryautomated multi-module cell processing instrument comprising a solidwall singulation/growth/editing/normalization module.

FIG. 10 is a simplified block diagram of an alternative embodiment of anexemplary automated multi-module cell processing instrument comprising asolid wall singulation/growth/editing/normalization module, in thiscase, used for recursive editing.

FIG. 11 is a simplified process diagram of yet another embodiment of anexemplary automated multi-module cell processing instrument, in thiscase without a singulation module.

FIG. 12A depicts an FTEP with a constricted flow channel withobstructions at the ends of the flow channel proximate to the inlet andoutlet (at right), and an obstruction array as described herein (atleft). FIG. 12B-12D are bar graphs depicting the number of cells thatwere input (far left bar), the number of cells that survivedelectroporation (left bar for each datapoint), and the number of cellsthat were transformed (right bar for each datapoint) at varying kV andpressures. The obstruction arrays described herein were benchmarkedagainst a NEPA device and a single constriction device.

FIG. 13 shows two graphs, at left shows simulated electric fieldstrength experienced by cells vs. time for cells passing through asingle constriction FTEP (such as shown in FIG. 12A, top) and at rightshows simulated electric field strength experienced by cells vs. timefor cells passing through a parallel-constriction FTEP (such as shown inFIGS. 1M and 1N).

FIG. 14 shows the results of electric field strength and residence timesweeps for the parallel-constriction FTEP as shown in FIG. 1L-1N wherethe obstructions in the center region define a 1 mm length, whichconfirms optimal uptake at 6.8 psi, 3 kV.

FIG. 15 shows the results of electric field strength and residence timesweeps for the parallel-constriction FTEP as shown in FIG. 1L-1N wherethe obstructions in the center region define a 0.5 mm length. Note thatthe parallel-constriction FTEP allows one to control the magnitude of aconstant, high electric field strength and the amount of time cells areexposed to the electric field by simply adjusting the applied voltageand pressure.

It should be understood that the drawings are not necessarily to scale,and that like reference numbers refer to like features.

DETAILED DESCRIPTION

All of the functionalities described in connection with one embodimentof the methods, devices or instruments described herein are intended tobe applicable to the additional embodiments of the methods, devices andinstruments described herein except where expressly stated or where thefeature or function is incompatible with the additional embodiments. Forexample, where a given feature or function is expressly described inconnection with one embodiment but not expressly mentioned in connectionwith an alternative embodiment, it should be understood that the featureor function may be deployed, utilized, or implemented in connection withthe alternative embodiment unless the feature or function isincompatible with the alternative embodiment.

The practice of the techniques described herein may employ, unlessotherwise indicated, conventional techniques and descriptions ofmolecular biology (including recombinant techniques), cell biology,biochemistry, and genetic engineering technology, which are within theskill of those who practice in the art. Such conventional techniques anddescriptions can be found in standard laboratory manuals such as Greenand Sambrook, Molecular Cloning: A Laboratory Manual. 4th, ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (2014);Current Protocols in Molecular Biology, Ausubel, et al. eds., (2017);Neumann, et al., Electroporation and Electrofusion in Cell Biology,Plenum Press, New York, 1989; and Chang, et al., Guide toElectroporation and Electrofusion, Academic Press, California (1992),all of which are herein incorporated in their entirety by reference forall purposes. Nucleic acid-guided nuclease techniques can be found in,e.g., Genome Editing and Engineering from TALENs and CRISPRs toMolecular Surgery, Appasani and Church (2018); and CRISPR: Methods andProtocols, Lindgren and Charpentier (2015); both of which are hereinincorporated in their entirety by reference for all purposes.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a cell” refers toone or more cells, and reference to “the system” includes reference toequivalent steps, methods and devices known to those skilled in the art,and so forth. Additionally, it is to be understood that terms such as“left,” “right,” “top,” “bottom,” “front,” “rear,” “side,” “height,”“length,” “width,” “upper,” “lower,” “interior,” “exterior,” “inner,”and/or “outer” that may be used herein merely describe points ofreference and do not necessarily limit embodiments of the presentdisclosure to any particular orientation or configuration. Furthermore,terms such as “first,” “second,” “third,” etc., merely identify one of anumber of portions, components, steps, operations, functions, and/orpoints of reference as disclosed herein, and likewise do not necessarilylimit embodiments of the present disclosure to any particularconfiguration or orientation.

Additionally, the terms “approximately,” “proximate,” “minor,” andsimilar terms generally refer to ranges that include the identifiedvalue within a margin of 20%, 10% or preferably 5% in certainembodiments, and any values therebetween.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated by reference for the purpose of describing anddisclosing devices, formulations and methodologies that may be used inconnection with the presently described invention.

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included in smaller ranges, and arealso encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, features and procedures well known to thoseskilled in the art have not been described in order to avoid obscuringthe invention. The terms used herein are intended to have the plain andordinary meaning as understood by those of ordinary skill in the art.

The term “complementary” as used herein refers to Watson-Crick basepairing between nucleotides and specifically refers to nucleotideshydrogen-bonded to one another with thymine or uracil residues linked toadenine residues by two hydrogen bonds and cytosine and guanine residueslinked by three hydrogen bonds. In general, a nucleic acid includes anucleotide sequence described as having a “percent complementarity” or“percent homology” to a specified second nucleotide sequence. Forexample, a nucleotide sequence may have 80%, 90%, or 100%complementarity to a specified second nucleotide sequence, indicatingthat 8 of 10, 9 of 10 or 10 of 10 nucleotides of a sequence arecomplementary to the specified second nucleotide sequence. For instance,the nucleotide sequence 3′-TCGA-5′ is 100% complementary to thenucleotide sequence 5′-AGCT-3′; and the nucleotide sequence 3′-TCGA-5′is 100% complementary to a region of the nucleotide sequence5′-TAGCTG-3′.

The term DNA “control sequences” refers collectively to promotersequences, polyadenylation signals, transcription termination sequences,upstream regulatory domains, origins of replication, internal ribosomeentry sites, nuclear localization sequences, enhancers, and the like,which collectively provide for the replication, transcription andtranslation of a coding sequence in a recipient cell. Not all of thesetypes of control sequences need to be present so long as a selectedcoding sequence is capable of being replicated, transcribed and—for somecomponents-translated in an appropriate host cell.

As used herein the term “donor DNA” or “donor nucleic acid” refers tonucleic acid that is designed to introduce a DNA sequence modification(insertion, deletion, substitution) into a locus by homologousrecombination using nucleic acid-guided nucleases. For homology-directedrepair, the donor DNA must have sufficient homology to the regionsflanking the “cut site” or site to be edited in the genomic targetsequence. The length of the homology arm(s) will depend on, e.g., thetype and size of the modification being made. In many instances andpreferably, the donor DNA will have two regions of sequence homology(e.g., two homology arms) to the genomic target locus. Preferably, an“insert” region or “DNA sequence modification” region—the nucleic acidmodification that one desires to be introduced into a genome targetlocus in a cell-will be located between two regions of homology. The DNAsequence modification may change one or more bases of the target genomicDNA sequence at one specific site or multiple specific sites. A changemay include changing 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 50, 75,100, 150, 200, 300, 400, or 500 or more base pairs of the targetsequence. A deletion or insertion may be a deletion or insertion of 1,2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 150, 200, 300, 400, or500 or more base pairs of the target sequence.

As used herein, “enrichment” refers to enriching for edited cells bysingulation, optionally inducing editing, and growth of singulated orsubstantially singulated cells into terminal-sized colonies (e.g.,saturation or normalization of colony growth). Alternatively,“enrichment” may be performed on a bulk liquid culture, by inducingediting when the cells are at the end of the logarithmic stage of growthor just after the cells enter growth senescence. Inducing editingentails inducing transcription of the nuclease, gRNA or both.

The terms “guide nucleic acid” or “guide RNA” or “gRNA” refer to apolynucleotide comprising 1) a guide sequence capable of hybridizing toa genomic target locus, and 2) a scaffold sequence capable ofinteracting or complexing with a nucleic acid-guided nuclease.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or, more often in the context of the presentdisclosure, between two nucleic acid molecules. The term “homologousregion” or “homology arm” refers to a region on the donor DNA with acertain degree of homology with the target genomic DNA sequence.Homology can be determined by comparing a position in each sequencewhich may be aligned for purposes of comparison. When a position in thecompared sequence is occupied by the same base or amino acid, then themolecules are homologous at that position. A degree of homology betweensequences is a function of the number of matching or homologouspositions shared by the sequences.

“Operably linked” refers to an arrangement of elements where thecomponents so described are configured so as to perform their usualfunction. Thus, control sequences operably linked to a coding sequenceare capable of effecting the transcription, and in some cases, thetranslation, of a coding sequence. The control sequences need not becontiguous with the coding sequence so long as they function to directthe expression of the coding sequence. Thus, for example, interveninguntranslated yet transcribed sequences can be present between a promotersequence and the coding sequence and the promoter sequence can still beconsidered “operably linked” to the coding sequence. In fact, suchsequences need not reside on the same contiguous DNA molecule (i.e.chromosome) and may still have interactions resulting in alteredregulation.

A “promoter” or “promoter sequence” is a DNA regulatory region capableof binding RNA polymerase and initiating transcription of apolynucleotide or polypeptide coding sequence such as messenger RNA,ribosomal RNA, small nuclear or nucleolar RNA, guide RNA, or any kind ofRNA transcribed by any class of any RNA polymerase I, II or III.Promoters may be constitutive or inducible, and in someembodiments-particularly many embodiments in which enrichment isemployed—the transcription of at least one component of the nucleicacid-guided nuclease editing system is under the control of an induciblepromoter.

As used herein the term “selectable marker” refers to a gene introducedinto a cell, which confers a trait suitable for artificial selection.General use selectable markers are well-known to those of ordinary skillin the art. Drug selectable markers such as ampicillin/carbenicillin,kanamycin, chloramphenicol, erythromycin, tetracycline, gentamicin,bleomycin, streptomycin, rifampicin, puromycin, hygromycin, blasticidin,and G418 may be employed. In other embodiments, selectable markersinclude, but are not limited to sugars such as rhamnose, human nervegrowth factor receptor (detected with a MAb, such as described in U.S.Pat. No. 6,365,373); truncated human growth factor receptor (detectedwith MAb); mutant human dihydrofolate reductase (DHFR; fluorescent MTXsubstrate available); secreted alkaline phosphatase (SEAP; fluorescentsubstrate available); human thymidylate synthase (TS; confers resistanceto anti-cancer agent fluorodeoxyuridine); human glutathioneS-transferase alpha (GSTA1; conjugates glutathione to the stem cellselective alkylator busulfan; chemoprotective selectable marker inCD34+cells); CD24 cell surface antigen in hematopoietic stem cells;human CAD gene to confer resistance to N-phosphonacetyl-L-aspartate(PALA); human multi-drug resistance-1 (MDR-1; P-glycoprotein surfaceprotein selectable by increased drug resistance or enriched by FACS);human CD25 (IL-2α; detectable by Mab-FITC); Methylguanine-DNAmethyltransferase (MGMT; selectable by carmustine); and Cytidinedeaminase (CD; selectable by Ara-C). “Selective medium” as used hereinrefers to cell growth medium to which has been added a chemical compoundor biological moiety that selects for or against selectable markers.

The terms “target genomic DNA sequence”, “target sequence”, or “genomictarget locus” refer to any locus in vitro or in vivo, in a nucleic acid(e.g., genome) of a cell or population of cells, in which a change of atleast one nucleotide is desired using a nucleic acid-guided nucleaseediting system. The target sequence can be a genomic locus orextrachromosomal locus.

A “vector” is any of a variety of nucleic acids that comprise a desiredsequence or sequences to be delivered to and/or expressed in a cell.Vectors are typically composed of DNA, although RNA vectors are alsoavailable. Vectors include, but are not limited to, plasmids, fosmids,phagemids, virus genomes, YACs, BACs, synthetic chromosomes, and thelike. As used herein, the phrase “engine vector” comprises a codingsequence for a nuclease to be used in the nucleic acid-guided nucleasesystems and methods of the present disclosure. The engine vector mayalso comprise, in a bacterial system, the λ Red recombineering system oran equivalent thereto. Engine vectors also typically comprise aselectable marker. As used herein the phrase “editing vector” comprisesa donor nucleic acid, optionally including an alteration to the targetsequence that prevents nuclease binding at a PAM or spacer in the targetsequence after editing has taken place, and a coding sequence for agRNA. The editing vector may also comprise a selectable marker and/or abarcode. In some embodiments, the engine vector and editing vector maybe combined; that is, all editing and selection components may be foundon a single vector. Further, the engine and editing vectors comprisecontrol sequences operably linked to, e.g., the nuclease codingsequence, recombineering system coding sequences (if present), donornucleic acid, guide nucleic acid, and selectable marker(s).

The Invention Generally

Electroporation is a widely-used method for permeabilization of cellmembranes that works by temporarily generating pores in the cellmembranes with electrical stimulation. The applications ofelectroporation include the delivery of DNA, RNA, siRNA, peptides,proteins, antibodies, drugs or other substances to a variety of cellssuch as mammalian cells (including human cells), plant cells, archaea,yeasts, other eukaryotic cells, bacteria, and other cell types. Further,mixtures of cell types can also be electroporated in a single run; e.g.,mixtures of E. coli strains, mixtures of bacterial strains, mixtures ofyeast strains, mixtures of mammalian cells. Electrical stimulation mayalso be used for cell fusion in the production of hybridomas or otherfused cells. During a typical electroporation procedure, cells aresuspended in a buffer or medium that is favorable for cell survival. Forbacterial cell electroporation, low conductance mediums, such as water,glycerol solutions and the like, are often used to reduce the heatproduction by transient high current. The cells and material to beelectroporated into the cells (collectively “the cell sample”) is thenplaced in a cuvette embedded with two flat electrodes for an electricaldischarge. For example, Bio-Rad (Hercules, Calif.) makes the GENE PULSERXCELL™ line of products to electroporate cells in cuvettes.Traditionally, electroporation requires high field strength.

Generally speaking, microfluidic flow-through electroporation(FTEP)—using cell suspension volumes of less than approximately 10 mland as low as 1 μl—allows for more precise control over the transfectionor transformation process and permits flexible integration with othercell processing tools compared to bench-scale electroporation devices.Microfluidic flow-through electroporation thus provides uniqueadvantages for, e.g., single cell transformation, processing, andanalysis; microfluidic electroporation may be used for multi-unit FTEPdevice configurations; and microfluidic electroporation devices may beintegrated into automated multi-module cell processing instruments.

A particular characteristic of the FTEP devices disclosed herein is thatrather than having a single flow path for the cells to be porated, theFTEP devices described herein have a single flow channel but many flowpaths. The flow channel comprises obstructions or flow diverters suchthat the single flow channel is separated or parallelized into many flowpaths. Configuring the flow channel into many flow paths provides theadvantage that if one flow path becomes clogged, there are several tomany alternative flow paths that may be taken. That is, if only a singleflow path is present and this single flow path is clogged or obstructed,the result is a catastrophic failure of the electroporation device.Further, at constant applied voltage, electric field strength can beincreased by reducing the spacing between the obstructions in theobstruction array. Thus, as the spacing between obstructions getssmaller, the electric field increases and thus the efficiency ofelectroporation of the cells increases.

The present disclosure provides FTEP devices, automated multi-moduleinstruments and methods of using FTEP devices that achieve highefficiency cell electroporation with low toxicity where theelectroporation devices and systems can be integrated with otherautomated cell processing tools. Further, the electroporation device ofthe disclosure allows for multiplexing where two to many electroporationunits are constructed and used in parallel, which allows forparticularly easy integration with robotic liquid handlinginstrumentation. Such automated instrumentation includes, but is notlimited to, off-the-shelf automated liquid handling systems from Tecan(Mannedorf, Switzerland), Hamilton (Reno, Nev.), Beckman Coulter (FortCollins, Colo.), etc.

During the electroporation process, it is important to use voltagesufficient for achieving electroporation of material into the cells, butnot too much voltage as too much power will decrease cell viability. Forexample, to electroporate a suspension of a human cell line, 200 voltsis needed for a 0.2 ml sample in a 4 mm-gap cuvette with exponentialdischarge from a capacitor of about 1000 μF. However, if the same 0.2 mlcell suspension is placed in a longer container with 2 cm electrodedistance (5 times of cuvette gap distance), the voltage required wouldbe 1000 volts, but a capacitor of only 40 μF ( 1/25 of 1000 μF) isneeded because the electric energy from a capacitor follows the equationof:E=0.5U ² Cwhere E is electric energy, U is voltage and C is capacitance.Therefore, a high voltage pulse generator is easy to manufacture becauseit needs a much smaller capacitor to store a similar amount of energy.Similarly, it would not be difficult to generate other wave forms ofhigher voltages.

The electroporation devices of the disclosure allow for a high rate ofcell transformation in a relatively short amount of time. The rate ofcell transformation is dependent on the cell type and the number ofcells being transformed. For example, for E. coli, the electroporationdevices can provide a cell transformation rate of 10³ to 10¹² cells perminute, 10⁴ to 10¹⁰ per minute, 10⁵ to 10⁹ per minute, or 10⁶ to 10⁸ perminute. Typically, 10⁷ to 10⁸ yeast cells are subjected totransformation and 10⁴ to 10⁵ are transformed per round oftransformation, and 10⁹-10¹⁰ bacterial are subjected to transformationand 10⁶ to 10⁷ are transformed per round of transformation. Theelectroporation devices also allow transformation of batches of cellsranging from 1 cell to 10¹¹ cells in a single transformation procedureusing parallel devices.

Exemplary FTEP Embodiments

An FTEP assembly is illustrated in FIGS. 1A-1C. FIGS. 1A through 1C aretop perspective, bottom perspective, and bottom views, respectively, ofan FTEP assembly 1500 comprising six co-joined FTEP devices 150. FIG. 1Adepicts six FTEP units 150 arranged on a single, integrally-formedinjection molded substrate 156. Each of the six FTEP units 150 havewells 152 that define inlets and wells 154 that define outlets. Further,on each FTEP unit one of two electrode channels 178 can be seen. FIG. 1Bis a bottom perspective view of the FTEP assembly 1500 with the sixco-joined FTEP devices 150 of FIG. 15A arranged on a single substrate156. Six inlet wells 152 can be seen, one for each flow-throughelectroporation unit 150, and one outlet well 154 can be seen on theleft-most FTEP unit. Also seen in FIG. 1B for each FTEP unit 150 are aninlet 102, an outlet 104, a flow channel 106 comprising five regions: aninlet-filter region 106 a, an inlet-proximal region 106 b, a centralregion 106 c, an outlet-proximal region 106 d, and an outlet-filterregion 106 e (only central region 106 c is labeled in this FIG. 1B, butsee FIG. 1C). Each FTEP unit further comprises two electrodes 108flanking central region 106 c of flow channel 106. Central region 106 cof flow channel 106 comprises a central portion (not labeled) comprisingan obstruction array 176 that includes a plurality of obstructions (notseen clearly in this FIG. 1B) which provide several to many paths forcells to travel through flow channel 106. Additionally seen are filters172 and 170 included in the inlet-filter region 106 a and outlet-filterregion 106 e, respectively, of flow channel 106 to prevent clogging inflow channel 106.

FIG. 1C is a bottom view of the FTEP assembly 1500 of the six co-joinedFTEP devices 150 of FIGS. 1A and 1B. Depicted in FIG. 1C are six FTEPunits 150 arranged on a single substrate 156, where each FTEP unit 150comprises an inlet 102, an outlet 104, a flow channel 106 comprisingfive regions: an inlet-filter region 106 a, an inlet-proximal region 106b, a central region 106 c, an outlet-proximal region 106 d, and anoutlet-filter region 106 e. Each FTEP unit further comprises twoelectrodes 108 flanking the central region 106 c of flow channel 106.Central region 106 c of flow channel 106 comprises a central portioncomprising an obstruction array 176 that includes a plurality ofobstructions (not seen clearly in this FIG. 1C) which provide a numberof flow paths for cells travelling through flow channel 106.Additionally seen are filters 172 and 170 included in the inlet-filterregion 106 a and outlet-filter region 106 e, respectively, of flowchannel 106 to prevent clogging of the channel. Once the six FTEP units150 are fabricated, they can be separated from one another (e.g.,“snapped apart”) upon the depicted score lines and used one at a time;alternatively, the FTEP units may be used in embodiments where two ormore FTEP units 150 are used in parallel.

The FTEP described herein comprises a filter 172 in the inlet-filterregion 106 a of flow channel 106 and a filter 170 in the outlet-filterregion 106 e of flow channel 106, as well as the obstruction array 176comprising a plurality of obstructions. The filters serve the purpose offiltering the fluid containing the cells and DNA (or other material tobe porated into the cells) before the fluid encounters either theinlet-proximal region 106 b or outlet-proximal region 106 d of flowchannel 106. In this embodiment, there are filters both at theinlet-filter region 106 a and outlet-filter region 106 e of flow channel106 because the FTEP devices may utilize a push-pull pneumatic means toflow liquids from inlet 102 to outlet 104, then from outlet 104 back toinlet 102 for another round of electroporation. The filter, like theobstruction array, decreases the likelihood that cells or other matterwill clog the flow channel. Instead, if there is particulate matter thatposes a threat to clogging the flow channel, the filter will catch theparticulate matter leaving other pores or flow paths through which therest of the cell/DNA/fluid can pass. Note that in this embodiment, thefilter has a gradient flow path size, from large flow paths at the inletto smaller flow paths toward the inlet proximal region 106 b of the flowchannel 106, and from large flow paths at the outlet 104 to small flowpaths toward the outlet-proximal region 106 d of flow channel 106;however, in alternative embodiments the flow paths may be the same size(e.g., not gradiated) or have an alternative gradient configuration.

The obstruction array 176 provides a number of flow paths for the cellsand the material to be porated into the cells that travel through flowchannel 106. Such a configuration reduces the likelihood of catastrophicfailure of the FTEP device from clogging compared to devices with asingle flow path. If one of the flow paths becomes clogged, the cellsand material to be porated into the cells can flow through analternative flow path. Again, a particular characteristic of the FTEPdevices disclosed herein is that rather than having a single flow pathfor the cells to be porated, the FTEP devices have a single flow channelwith many flow paths. In the devices described herein, the flow channelcomprises obstructions of flow diverters such that the flow channel isseparated or parallelized into many flow paths. Configuring the flowchannel into many flow paths provides the advantage that if one flowpath becomes clogged, there are several to many alternative flow pathsthat may be taken. That is, if only a single flow path is present andthis single flow path is clogged or obstructed, the result is acatastrophic failure of the electroporation device.

The substrate, inlet wells, outlet wells, filters and obstruction arraysof the FTEP device can be made from many materials depending on whetherthe FTEP device is to be reused, autoclaved, or is disposable, includingstainless steel, silicon, glass, resin, polyvinyl chloride,polyethylene, polyamide, polystyrene, polyethylene, polypropylene,acrylonitrile butadiene, polycarbonate, polyetheretheketone (PEEK),polysulfone and polyurethane, co-polymers of these and other polymers.Similarly, the walls of the channels in the device can be made of anysuitable material including silicone, resin, glass, glass fiber,polyvinyl chloride, polyethylene, polyamide, polyethylene,polypropylene, acrylonitrile butadiene, polycarbonate,polyetheretheketone (PEEK), polysulfone and polyurethane, co-polymers ofthese and other polymers. Preferred materials include crystal styrene,cyclo-olefin polymer (COP) and cyclic olefin co-polymers (COC), whichallow the FTEP device to be formed entirely by injection molding in onepiece with the exception of the electrodes and, e.g., a bottom and/ortop sealing film if present.

The FTEP devices described herein (or portions of the FTEP devices) canbe created or fabricated via various techniques, e.g., as entire devicesor by creation of structural layers that are fused or otherwise coupled.For example, for metal FTEP devices, fabrication may include precisionmechanical machining or laser machining; for silicon FTEP devices,fabrication may include dry or wet etching; for glass FTEP devices,fabrication may include dry or wet etching, powderblasting,sandblasting, or photostructuring; and for plastic FTEP devices,fabrication may include thermoforming, injection molding, hot embossing,or laser machining. The components of the FTEP devices may bemanufactured separately and then assembled, or certain components of theFTEP devices (or even the entire FTEP device except for the electrodes)may be manufactured (e.g., using 3D printing) or molded (e.g., usinginjection molding) as a single entity, with other components added aftermolding. For example, housing and channels may be manufactured or moldedas a single entity, with the electrodes later added to form the FTEPunit. In some embodiments, a film or a flat substrate may be used toseal the bottom of the device. The film, in some embodiments, is madefrom the same material as the FTEP device, in this case, e.g., crystalstyrene, cyclo-olefin polymer (COP) or cyclic olefin co-polymers (COC).The FTEP device may also be formed in two or more parallel layers, e.g.,a layer with the horizontal channel and filter, a layer with thevertical channels, and a layer with the inlet and outlet ports, whichare manufactured and/or molded individually and assembled followingmanufacture.

In specific aspects, the FTEP device can be manufactured using a circuitboard as a base, with the electrodes, filter and/or the flow channelformed in the desired configuration on the circuit board, and theremaining housing of the device containing, e.g., the one or more inletand outlet channels and/or the flow channel formed as a separate layerthat is then sealed onto the circuit board. The sealing of the top ofthe housing onto the circuit board provides the desired configuration ofthe different elements of the FTEP devices of the disclosure. Also, twoto many FTEP devices (up to 48 or more) may be manufactured in parallelon a single substrate, then separated from one another thereafter orused in parallel. In certain embodiments, the FTEP devices are reusableand, in some embodiments, the FTEP devices are disposable. In additionalembodiments, the FTEP devices may be autoclavable.

The electrodes 108 can be formed from any suitable metal, such ascopper, stainless steel, titanium, aluminum, brass, silver, rhodium,gold or platinum, or graphite. One preferred electrode material is alloy303 (UNS330300) austenitic stainless steel. An applied electric fieldcan destroy electrodes made from of metals like aluminum. If amultiple-use (e.g., non-disposable) FTEP device is desired—as opposed toa disposable, one-use FTEP device—the electrode plates can be coatedwith metals resistant to electrochemical corrosion. Conductive coatingslike noble metals, e.g., gold, can be used to protect the electrodeplates.

The overall size of the FTEP device may be from 3 cm to 15 cm in length,or 4 cm to 12 cm in length, or 4.5 cm to 10 cm in length. The overallwidth of the FTEP device may be from 0.5 cm to 5 cm, or from 0.75 cm to3 cm, or from 1 cm to 2.5 cm, or from 1 cm to 1.5 cm.

In embodiments of the FTEP device where reservoirs are used to introducecells and exogenous material into the FTEP device, the reservoirs rangein volume from 100 μL to 10 mL, or from 500 μL to 7.5 mL, or from 1 mLto 5 mL. The flow rate in the FTEP ranges from 0.01 mL to 5.0 mL perminute, or from 0.05 mL to 3.0 mL per minute, or from 0.1 mL to 2.5 mLper minute or from 0.2 to 2.0 mL per minute. The pressure in the FTEPdevice ranges from 1-30 psi, or from 2-10 psi, or from 3-5 psi.

To avoid different field intensities between the electrodes, theelectrodes should be arranged in parallel. Furthermore, the surface ofthe electrodes should be as smooth as possible without pin holes orpeaks. Electrodes having a roughness Rz of 1 to m are preferred. Inanother embodiment of the invention, the flow-through electroporationdevice comprises at least one additional electrode which applies aground potential to the FTEP device.

The electrodes are configured to deliver 1-50 kV/cm, or 5-40 kV/cm, or10-25 kV/cm. The further apart the electrodes are, the more voltageneeds to be supplied; in addition, the voltage delivered of coursedepends on the types of cells being porated, the medium in which thecells are suspended, the size of the electroporation channel, and thelength and diameter of the electrodes. There are many different pulseforms that may be employed with the FTEP device, including exponentialdecay waves, square or rectangular waves, arbitrary wave forms, or aselected combination of wave forms. One type of common pulse form is theexponential decay wave, typically made by discharging a loaded capacitorto the cell sample. The exponential decay wave can be made less steep bylinking an inductor to the cell sample so that the initial peak currentcan be attenuated. When multiple waveforms in a specified sequence areused, they can be in the same direction (direct current) or differentdirections (alternating current). Using alternating current can bebeneficial in that two topical surfaces of a cell instead of just onecan be used for molecular transport, and alternating current can preventelectrolysis. The pulse generator can be controlled by a digital oranalog panel. In some embodiments, square wave forms are preferred, andin other embodiments, an initial wave spike before the square wave ispreferred.

The FTEP device may be configured to electroporate cell sample volumesbetween 1 μL to 5 ml, 25 μL to 2.5 ml, 50 μL to 2 ml, 100 μL to 1 μL, or200 μL to 750 L. The medium or buffer used to suspend the cells andmaterial (reagent) to be electroporated into the cells for theelectroporation process may be any suitable medium or buffer for thetype of cells being transformed or transfected, such as SOC, MEM, DMEM,IMDM, RPMI, Hanks', PBS and Ringer's solution, where the media may beprovided, e.g., in a reagent cartridge as part of a kit. Further,because the cells must be made electrocompetent prior to transformationor transfection, the buffer also may comprise glycerol or sorbitol, andmay also comprise a surfactant. For electroporation of most eukaryoticcells the medium or buffer usually contains salts to maintain a properosmotic pressure. The salts in the medium or buffer also render themedium conductive. For electroporation of very small prokaryotic cellssuch as bacteria, sometimes water or 10% glycerol is used as a lowconductance medium to allow a very high electric field strength. In thatcase, the charged molecules to be delivered still render water-basedmedium more conductive than the lipid-based cell membranes and themedium may still be roughly considered as conductive particularly incomparison to cell membranes.

The compound to be electroporated into the cells can be any compoundknown in the art to be useful for electroporation, such as nucleicacids, oligonucleotides, polynucleotides, DNA, RNA, peptides, proteinsand small molecules like hormones, cytokines, chemokines, drugs, or drugprecursors. In addition, the FTEP devices may comprise push-pullpneumatic means to allow multi-pass electroporation procedures; that is,cells to be electroporated may be “pulled” from the inlet toward theoutlet for one pass of electroporation, then be “pushed” from the outletend of the flow-through FTEP device toward the inlet end to pass betweenthe electrodes again for another pass of electroporation. This processmay be repeated one to many times. Alternatively, the FTEP may be usedto porate sequential aliquots of cells; for example, a first volume ofcells is porated in a first pass with the first volume then transferredto recovery, then a second volume of cells is porated in a second passwith the second volume then transferred to recovery, and so on withthird, fourth and fifth volumes or more.

FIG. 1D is an enlarged bottom view of an FTEP device 150 with theregions of the flow channel labeled. The FTEP device 150 comprises aninlet 102, an outlet 104, a flow channel 106 comprising five regions: aninlet-filter region 106 a, an inlet-proximal region 106 b, a centralregion 106 c, an outlet-proximal region 106 d, and an outlet-filterregion 106 e. Two electrodes 108 flank the central region 106 c of flowchannel 106 which comprises the obstruction array. Also seen are ramps174 a and 174 b. Ramp 174 a proximal to inlet 102 decreases thecross-sectional area of flow channel 106 from the region of ramp 174 aproximal to electrode 108 traveling toward the region of ramp 174 aproximal to the central region 106 c of flow channel 106. Ramp 174 bproximal to outlet 104 increases the cross-sectional area of flowchannel 106 from the region of ramp 174 b proximal to central region 106c of flow channel 106 traveling toward electrode 108. Channel height isa parameter that can be used to tune electric field strength. Atconstant applied voltage, the electric field strength can be increasedby reducing the cross-sectional area of the flow channel through whichthe cells pass. For example, as the height of the flow channeldecreases, the electric field strength increases. Similarly, asdescribed above, when the spacing between the obstructions in theobstruction array gets smaller, the electric field strength increases.Thus, the optional ramps serve the purpose of increasing electric fieldstrength to achieve enhanced electroporation efficiency. Ramps 174 a and174 b may be configured similarly (though in mirror image) or may havedifferent configurations. The ramps can range in length from 0.3 mm to4.0 mm, or from 0.5 mm to 3.0 mm, or from 0.8 mm to 2.4 mm. Width W ofthe ramp 174 is preferably equal to that of the channel, such asapproximately 0.5 mm to 10 mm, or from 1 mm to 5 mm, or from 1.5 mm to 4mm. Ramp 174 a decreases the cross-sectional height of central region106 c of flow channel 106—and ramp 174 b increases the cross-sectionalheight of central region 106 c of flow channel 106 to electrode 108—from250 μm to 25 μm, or from 200 μm to 50 μm, or from 100 μm to 25 μm.Additionally, the configuration of ramps 174 a and 174 b may be a smoothtransition of flow channel height from larger cross-sectional height tosmaller cross-sectional height, or the configuration of ramps 174 a and174 b may be stepped. For example for ramp 174 a, a first step maydecrease the cross-sectional height of central region 106 c by 25 mm fora length X of central region 106 c, then a next step may decrease thecross-sectional height of central region 106 c by another 25 mm forlength Y of central region 106 c. Again, the configuration of ramp 174 bmay match (mirror image) the configuration of ramp 174 a or may bedifferent than that of 174 a.

Obstruction array 176 includes a plurality (e.g., several to many) ofobstructions (not seen clearly in this FIG. 1D) which provide a numberof flow paths for cells to travel through flow channel 106. Additionallyseen are filters 172 and 170 included in the inlet-filter region 106 aand outlet-filter region 106 e, respectively, of flow channel 106 toprevent clogging of flow channel 106. FIG. 1E is a blow up of thecircled portion 182 of FIG. 1D showing detail of central region 106 c offlow channel 106. Seen in FIG. 1E are two electrodes 108, disposed inelectrode channels 178, which flank central region 106 c of flow channel106. The detail of obstruction array 176 is seen, with individualobstructions 180. In addition, optional ramp regions 174 a and 174 b areseen. The ramp regions ramp up (e.g. creates vertical cross-sectionalnarrowing of flow channel 106) from the inlet-proximal region 106 b (notseen) of flow channel 106 toward central region 106 c, and ramp down(e.g., creates a vertical widening of flow channel 106) from centralregion 106 c toward outlet proximal region 106 d (not shown) of flowchannel 106 as described above.

Depending on the type of cells to be electroporated (e.g., bacterial,yeast, mammalian) and the configuration of the electrodes, the distancebetween the electrodes in the flow channel can vary widely. The lengthL1 from the mid-point of each electrode 108 is approximately 1 to 15 mm,or 2 to 12 mm, 3 to 10 mm, or 4 mm to 8 mm.

FIG. 1F-1K show different views of obstruction arrays 176, withindividual obstructions 180. FIG. 1F depicts a top view of centralregion 106 c of the flow channel (not labeled), with a central portion110 of central region 106 c, ramps 174 a and 174 b, obstruction array176, and electrodes 108 disposed in electrode channels 178. FIG. 1G is atop perspective view of the central region (not labeled) of the flowchannel (also not labeled), depicting central portion 110 of the centralregion, obstruction array 176, ramps 174, and electrodes 108 disposedwithin electrode channels 178. FIG. 1H is a close up of an obstructionarray (not labeled) showing obstructions 180 and flow paths 182 betweenobstructions 180. Although the obstructions are shown in this embodimentas oval-shaped, flat-topped elements, it should be understood that theobstructions may be triangular-, square-, rectangular-, pentagonal-,hexagonal-, rounded peg-, elliptical-, elongated oval—(see, e.g., FIG.1L-1N), or other faceted-shaped elements, and may be a combination ofshapes and configurations of obstruction elements.

In this embodiment, there are 40 total obstructions, and the size of theflow paths moving from the inlet-proximal region of the flow channel(not labeled) to the center region of the central region 106 c of theflow channel decreases; that is, the number of obstructions in a rowmoving from the inlet-proximal region of the flow channel to the centerportion 110 of the central region 106 c of the flow channel increasesfrom 3 to 5 to 7 to 10. Similarly, the size of the flow paths movingfrom the center portion 110 of the central region 106 c to theoutlet-proximal region of the flow channel (not labeled) increases; thatis, the number of obstructions in a row moving from the center portion110 of the central region 106 c to the outlet-proximal region of theflow channel (not labeled) decreases from 10 to 7 to 5 to 3. At themid-point of the obstruction array (e.g., the center portion 110 ofcenter region of the flow path (not labeled) there are 10 obstructions180 defining 11 flow paths. The number of obstructions can range from 4to 150, from 6 to 120, from 8 to 100, or from 10 to 90. The pattern orconfiguration of the obstructions can be symmetrical (as in FIG. 1F-1K)or random. In this embodiment, the oval-shaped or elongated obstructionsare approximately 220 μm long and 100 μm wide, and the obstructions areat least about 100 μm apart, thus forming minimal flow path widths of100 μm. However, in alternative embodiments the obstructions can be from50 μm long to 20 μm wide, from 100 μm long to 50 μm wide, or from 250 μmlong to 150 μm wide. Round obstructions may be used instead or incombination with elongated obstructions or with other configurations ofobstructions. Round obstructions may be approximately between 25 μm-200μm in diameter, or between 50 μm to 150 μm in diameter, or 75 μm to 125μm in diameter. The flow paths or distance between obstructions canrange from 10 μm to 350 μm, from 20 μm to 300 μm, or from 30 μm to 250μm.

FIGS. 11-1K are similar to FIG. 1F-1H, showing views of analternately-configured obstruction array 176 and individual obstruction180 s. FIG. 11 shows a top view of central region 106 c of flow channel(not labeled), with central portion 110, ramps 174, and electrodes 108in electrode channels (not labeled). FIG. 1J is a top view of an area ofan obstruction array (not labeled) showing individual obstructions 180and central portion 110 of the central region (not labeled) of the flowchannel (also not labeled). Note that the outer margin of the FTEPdevice in central region (not labeled) coincident to the obstructionarray is contoured to provide flow paths 182 similar to the flowchannels between obstructions 180. FIG. 1K is a close-up view ofobstructions 180 and flow paths 182. Again the obstructions are shown inthis embodiment as oval-shaped, flat-topped elements; however, it shouldbe understood that the obstructions may be triangular-, square-,rectangular-, pentagonal-, hexagonal-, rounded peg-, elliptical-,elongated oval- or other faceted-shaped elements, and may be acombination of shapes and configurations of obstruction elements. Inthis embodiment, there are 33 total obstructions in a 3-5-5-7-5-5-3pattern moving from the inlet-proximal region of the flow channel to thecenter portion of the center region of the flow channel to theoutlet-proximal region of the flow channel (regions not labeled).

FIG. 1L is a bottom view of an alternative embodiment of an obstructionFTEP device 190, here a parallel-obstruction device, with the regions ofthe flow channel labeled. The parallel-obstruction FTEP device 190comprises an inlet 102, an outlet 104, a flow channel 106 comprisingfive regions: an inlet-filter region 106 a, an inlet-proximal region 106b, a central region 106 c, an outlet-proximal region 106 d, and anoutlet-filter region 106 e. Two electrodes 108 flank the central region106 c of flow channel 106 which comprises the obstruction array 177.Also seen are steps 174 c and 174 d. Steps 174 c and 174 d are much likeramps 174 a and 174 b seen in FIGS. 1D, 1E, 1G and 11; however, insteadof a “ramp” or gradual decreasing of channel height (with ramp 174 a) orgradual increasing of channel height (as with ramp 174 b), steps 174 cand 174 d are true steps—that is, the decrease or increase in channelheight to and from the central channel region 106 c is abrupt. Step 174c proximal to inlet 102 decreases the cross-sectional area of flowchannel 106 from the region of step 174 c proximal to electrode 108traveling toward the region of step 174 c proximal to the central region106 c of flow channel 106. Step 174 d proximal to outlet 104 increasesthe cross-sectional area of flow channel 106 from the region of step 174d proximal to central region 106 c of flow channel 106 traveling towardelectrode 108 and outlet 104. As described above, channel height is aparameter that can be used to tune electric field strength. At constantapplied voltage, the electric field strength can be increased byreducing the cross-sectional area of the flow channel through which thecells pass. For example, as the height of the flow channel decreases,the electric field strength increases. The steps serve the purpose ofincreasing electric field strength to achieve enhanced electroporationefficiency. As with ramps 174 a and 174 b, steps 174 c and 174 d may beconfigured similarly (though in mirror image) or may have differentconfigurations. For an exemplary channel height of 100 m, the step canrange in height from 10 μm to 80 μm, or from 20 μm to 70 μm, or from 20μm to 60 m. Width W of the steps 174 c and 174 d is preferably equal tothat of the channel, such as approximately 0.5 mm to 3.0 mm, or from 1.0mm to 2.0 mm, or from 1.25 mm to 1.75 mm. For other channel heights, thesteps would be of the same proportion. Again, the configuration of step174 c may match (mirror image) the configuration of step 174 d or may bedifferent than that of 174 d. Parallel-obstruction array 177 includes aplurality (e.g., several to many) of obstructions in central region 106c (not seen clearly in this FIG. 1L) which provide a number of flowpaths for cells to travel through flow channel 106. Additionally seenare filters 172 and 170 included in the inlet-filter region 106 a andoutlet-filter region 106 e, respectively, of flow channel 106 to preventclogging of flow channel 106.

FIGS. 1M and 1N show different views of parallel-obstruction arrays 177(e.g., obstructions with a different configuration than shown in FIGS.1D-1K), with individual obstructions 180. In FIGS. 1M and 1N, theobstructions or flow diverters are elongated oval structures that arearranged parallel to one another forming “lanes” for cell flow. That is,instead of a plurality of obstructions arranged in a pattern such as inrows (see, e.g., the 3-5-5-7-5-5-3 pattern in FIGS. 11-1K), the presentparallel-obstruction embodiment comprises elongated obstructions thatform flow “lanes.” FIG. 1M depicts a top view of central region 106 c ofthe flow channel (not labeled), with a central portion 110 of centralregion 106 c, steps 174 c and 174 d, obstruction array 177, andelectrodes 108 disposed in electrode channels 178. Note that the lengthof the obstruction array 177 in channel 106 is denoted here as 1.0 mm inlength with the length of the “shelf” formed by the steps approximately1.1 mm in length. FIG. 1N is a top perspective view of the centralregion (not labeled) of the flow channel (also not labeled), depictingparallel-obstruction array 177 in channel 106 (not labeled) and step 174c, obstructions 180 and flow paths 182 between obstructions 180.Although the obstructions are shown in this embodiment as longoval-shaped, flat-topped elements, it should be understood that theobstructions may be triangular-, square-, rectangular-, pentagonal-,hexagonal-, rounded peg-, elliptical- or other faceted-shaped elements,and may be a combination of shapes and configurations of obstructionelements. Note that step 174 c decreases the channel height in thisexemplary embodiment from 100 μm to 50 μm, and the height ofobstructions 180 is 50 μm.

In this embodiment, there are 7 total obstructions in parallel,resulting in 8 parallel paths for cell flow. In this embodiment, theelongated obstructions are approximately 1000 μm long and 100 μm wide,and the obstructions are at least about 200 μm apart center-to-center ofthe obstructions, thus forming minimal flow path widths of approximately100 μm. However, in alternative embodiments the obstructions can be from200 μm long to 25 μm wide, from 500 μm long to 50 μm wide, or from 4000μm long to 100 μm wide, with the length of the obstructions limited onlyby the distance between electrodes. The flow lanes between obstructionscan range from 2 μm to 300 μm, from 10 μm to 200 μm, or from 25 μm to100 μm in width.

FIG. 1O depicts (i) the electrodes 108 positioned on an electrodeplatform 158 before insertion into the FTEP array 1500; here an FTEParray comprising six FTEP devices (not labeled), where each FTEP devicehas an inlet well 152 and an outlet well 154. In use, the FTEP devicesare used in an orientation inverted relative to that shown in FIG. 1O(i). FIG. 1O (ii) depicts an electrode 108 contained within andprojecting from an electrode sheath 184. FIG. 1O (iii) depicts theelectrode 108 within electrode sheath 184 and inserted into an electrodechannel 178 with the electrode channel 178 (and electrode 108) adjacentto flow channel 106. In the embodiment shown in FIG. 1O (iii), electrode108 is even/flush with the wall of flow channel 106; that is, electrode108 is not in the path of the cells/DNA/fluid flowing through flowchannel 106; however, neither is the electrode recessed within theelectrode channel 178. In alternative embodiments, electrode 108 may berecessed within electrode channel 178. The configuration of theelectrode channel 178 and the flow channel 106 help prevent trapping airand reduce discontinuities in the electric field.

FIG. 1P shows scanning electron micrographs of filter 172. Note in thisembodiment, the porosity of filter 172 varies from large flow paths nearthe inlet 102 to small flow paths near inlet proximal region 106 b (notshown). When a second filter is present at the outlet-filter region 106e (not shown), the second filter may also vary in porosity. In the caseof a second filter between the outlet and the outlet-proximal region 106d, the filter can vary from large flow paths near the outlet proximalregion to small flow paths toward the outlet-proximal region 106 d.Scale information is shown in each micrograph. Moreover, as with theconfiguration of the obstructions, though the scanning electronmicrographs in FIG. 1P show the filter elements as rounded “pegs”, itshould be understood that the filter elements may be triangular-,square-, rectangular-, pentagonal-, hexagonal-, oval-, elliptical- orother faceted-shaped pegs, or a combination of differently-configuredand differently-sized filter elements.

Reagent Cartridges Comprising FTEPs

FIG. 2A depicts an exemplary combination reagent cartridge 200comprising an FTEP device 206 (“cartridge” or “reagent cartridge”) thatmay be used in an automated multi-module cell processing instrument.Cartridge 200 comprises a body 202, and reagent receptacles orreservoirs 204 along with an FTEP device 206 (exemplary embodiments ofwhich are described in detail in relation to FIGS. 1A-1P). Cartridge 200may be disposable or may be configured to be reused. Cartridge 200 maybe made from any suitable material, including stainless steel, aluminum,paper or other fiber, or plastics including polyvinyl chloride, cyclicolefin copolymer (COC), polyethylene, polyamide, polypropylene,acrylonitrile butadiene, polycarbonate, polyetheretheketone (PEEK),poly(methyl methylacrylate) (PMMA), polysulfone, and polyurethane, andco-polymers of these and other polymers. If the cartridge is disposable,preferably it is made of plastic or paper. Preferably the material usedto fabricate the cartridge is thermally-conductive, as in certainembodiments the cartridge 200 contacts a thermal device (not shown) thatheats or cools reagents in the reagent receptacles or reservoirs 204. Insome embodiments, the thermal device is a Peltier device orthermoelectric cooler. Reagent receptacles or reservoirs 204 may bereceptacles into which individual tubes of reagents are inserted asshown in FIG. 2A, receptacles into which one or more multiple co-joinedtubes are inserted (e.g., a row of four tubes that are co-joined areinserted into the reagent receptacles), or the reagent receptacles mayhold the reagents without inserted tubes with the reagents dispenseddirectly into the receptacles or reservoirs. Additionally, thereceptacles 204 in a reagent cartridge 200 may be configured for anycombination of tubes, co-joined tubes, and direct-fill of reagents.

In one embodiment, the reagent receptacles or reservoirs 204 of reagentcartridge 200 are configured to hold various size tubes, including,e.g., 250 ml tubes, 25 ml tubes, 10 ml tubes, 5 ml tubes, and Eppendorf(e.g., microcentrifuge) tubes. In yet another embodiment, allreceptacles may be configured to hold the same size tube, e.g., 5 mltubes, and reservoir inserts may be used to accommodate smaller tubes inthe reagent reservoir. In yet another embodiment-particularly in anembodiment where the reagent cartridge 200 is disposable—the reagentreservoirs 204 hold reagents without inserted tubes. In this disposableembodiment, the reagent cartridge may be part of a kit, where thereagent cartridge is pre-filled with reagents and the receptacles orreservoirs sealed with, e.g., foil, film, heat seal acrylic or the likeand presented to a consumer where the reagent cartridge can then be usedin an automated multi-module cell processing instrument. The reagentscontained in the reagent cartridge 200 will vary depending on work flow;that is, the reagents will vary depending on the processes to which thecells are subjected in the automated multi-module cell processinginstrument. For various embodiments of reagent cartridges of particularuse in automated multi-module cell processing instruments, see U.S. Pat.No. 10,376,889, issued 13 Aug. 2019; U.S. Pat. No. 10,406,525, issued 10Sep. 2019; and U.S. Pat. No. 10,478,822, issued 19 Nov. 2019.

FIG. 2B depicts an exemplary matrix configuration 240 for the reagentscontained in the reagent cartridges of FIG. 2A, where this matrixembodiment is a 4×4 reagent matrix. Through a matrix configuration, auser (or programmed processor) can locate the proper reagent for a givenprocess. That is, reagents such as cell samples, enzymes, buffers,nucleic acid vectors, expression cassettes, reaction components (suchas, e.g., MgCl₂, dNTPs, isothermal nucleic acid assembly reagents, GapRepair reagents, and the like), wash solutions, ethanol, and magneticbeads for nucleic acid purification and isolation, etc., are positionedin the matrix 240 at a known position. For example, reagents are locatedat positions A1 (210), A2 (211), A3 (212), A4 (213), B1 (214), B2 (215)and so on through, in this embodiment, to position D4 (225). FIG. 2A islabeled to show where several reservoirs 204 correspond to matrix 240;see receptacles 210, 211, 212, 213, 221 and 225. Although the reagentcartridge 200 of FIG. 2A and the matrix configuration 240 of FIG. 2Bshows a 4×4 matrix, matrices of the reagent cartridge andelectroporation devices can be any configuration, such as, e.g., 2×2,2×3, 2×4, 2×5, 2×6, 3×3, 3×5, 4×6, 6×7, or any other configuration,including asymmetric configurations, or two or more different matricesdepending on the reagents needed for the intended workflow.

In preferred embodiments of reagent cartridge 200 shown in FIG. 2A, thereagent cartridge comprises a script (not shown) readable by a processor(not shown) for dispensing the reagents via a liquid handling device(ADP head shown at 432 of FIG. 4A) and controlling the electroporationdevice contained within reagent cartridge 200. Also, the reagentcartridge 200 as one component in an automated multi-module cellprocessing instrument may comprise a script specifying two, three, four,five, ten or more processes performed by the automated multi-module cellprocessing instrument, or even specify all processes performed by theautomated multi-module cell processing instrument. In certainembodiments, the reagent cartridge is disposable and is pre-packagedwith reagents tailored to performing specific cell processing protocols,e.g., genome editing or protein production. Because the reagentcartridge contents vary while components of the automated multi-modulecell processing instrument may not, the script associated with aparticular reagent cartridge matches the reagents used and cellprocesses performed. Thus, e.g., reagent cartridges may be pre-packagedwith reagents for genome editing and a script that specifies the processsteps (or a script that modifies the steps of a pre-programmed scriptbased on, e.g., an updated reagent in the reagent cartridge) forperforming genome editing in an automated multi-module cell processinginstrument such as described in relation to FIGS. 4A-4C.

For example, the reagent cartridge 200 of FIG. 2A may comprise a scriptto pipette electrocompetent cells from reservoir A2 (211), transfer thecells to the electroporation device 206, pipette a nucleic acid solutioncomprising an editing vector from reservoir C3 (220), transfer thenucleic acid solution to the electroporation device, initiate theelectroporation process for a specified time, then move the poratedcells to a reservoir D4 (225) in the reagent cassette or to anothermodule such as the rotating growth vial (see, e.g., 418 of FIG. 4A) inthe automated multi-module cell processing instrument in FIG. 4A. Inanother example, the reagent cartridge may comprise a script to pipettetransfer of a nucleic acid solution comprising a vector from reservoirC3 (220), nucleic acid solution comprising editing oligonucleotidecassettes in reservoir C4 (221), and isothermal nucleic acid assemblyreaction mix from A1 (210) to an isothermal nucleic acidassembly/desalting reservoir. The script may also specify process stepsperformed by other modules in the automated multi-module cell processinginstrument. For example, the script may specify that the isothermalnucleic acid assembly/desalting module be heated to 50° C. for 30 min togenerate an assembled isothermal nucleic acid product; and desalting ofthe assembled isothermal nucleic acid product via magnetic bead-basednucleic acid purification involving a series of pipette transfers andmixing of magnetic beads in reservoir B2 (215), ethanol wash inreservoir B3 (216), and water in reservoir C1 (218) to the isothermalnucleic acid assembly/desalting reservoir (not seen in FIG. 4A).

FIGS. 3A-3C depict three side perspective views of a flow-throughelectroporation device insert 308 configured to be inserted into, e.g.,a reagent cartridge. In the embodiment of reagent cartridge 200 depictedin FIG. 2A, the flow-through electroporation device 206 (in FIGS. 3A-3F,FTEP 306) is located in the reagent cartridge 200 (also see reagentcartridge 410 with flow-through electroporation device 430 as onecomponent of an automated multi-module cell processing instrument 400 inFIG. 4A); although in alternative embodiments, the FTEP module may beseparate from the reagent cartridge. The FTEP comprises an inlet well352 (covered in FIGS. 3A and 3B), and outlet well 354 (also covered inFIGS. 3A and 3B), and the exterior of the electrode channels 378. TheFTEP device insert 308 comprises both a tab 317, and an outer flange307. FIG. 3C depicts the FTEP device insert 308 with a cover 305 for,e.g., shipping and to keep the FTEP device 306 sterile until use. TheFTEP inserts may be made of any appropriate material; however, theinserts are in most embodiments disposable, so typically are fabricatedfrom biocompatible plastics, including polyvinyl chloride, cyclic olefincopolymer (COC), polyethylene, polyamide, polypropylene, acrylonitrilebutadiene, polycarbonate, polyetheretheketone (PEEK), poly(methylmethylacrylate) (PMMA), polysulfone, and polyurethane, and co-polymersof these and other polymers.

FIGS. 3D-3F offer additional views of an FTEP insert 308. FIG. 3D is across section of the FTEP insert 308, housing FTEP 306 with inlet well352, outlet well 354, and electrode channels 378. FTEP insert 308comprises an outer flange 307, an FTEP cover 305, and tab 317, which isconfigured to engage with, e.g., a tab engagement member (not shown) ina reagent cartridge when inserted into a reagent cartridge. Also shownis FTEP cover 305, which in this embodiment is a tear-off foil, film orother type seal that is used to maintain the sterility of the FTEP untilready for use. FIG. 3E is a top view of the FTEP insert 308 shown inFIG. 3D. Seen are FTEP insert cover or seal 305, which protects andkeeps sterile the FTEP device before use and is removable by a user,data 373, and machine-readable indicia 375. Data 373 may includeinformation such as a lot number, a serial number, a product number, anexpiration date, or other data pertinent to FTEP insert 308.Machine-readable indicia 375 may be a barcode, QR code, a Data Matrixcode (error correction-type barcode), RFID or other type ofmachine-readable indicia, detected by one or more imaging sensors (e.g.,barcode scanners, cameras, etc.) (not shown) located in an automatedmulti-module cell processing instrument to, e.g., confirm the contentsof and optionally to control the operation of FTEP insert 308. FIG. 3Fis a top view of FTEP insert 308 with FTEP insert cover 305 (seen inFIG. 3E) removed. Again, data 373, machine-readable indicia 375, andFTEP 306 can be seen. Also, electrodes channels 378 of FTEP 306 areseen.

Nucleic Acid-Directed Nuclease Genome Editing Generally

The FTEP device of the present invention may be a stand-alone device ormodule or may be one module in an automated multi-module cell processinginstrument. In one embodiment, the FTEP device is used in an automatedcell processing instrument designed for creating genome edits in livecells. A recent discovery for editing live cells involves nucleicacid-guided nuclease (e.g., RNA-guided nuclease) editing. A nucleicacid-guided nuclease complexed with an appropriate synthetic guidenucleic acid in a cell can cut the genome of the cell at a desiredlocation. The guide nucleic acid helps the nucleic acid-guided nucleaserecognize and cut the DNA at a specific target sequence. By manipulatingthe nucleotide sequence of the guide nucleic acid, the nucleicacid-guided nuclease may be programmed to target any DNA sequence forcleavage as long as an appropriate protospacer adjacent motif (PAM) isnearby. In certain aspects, the nucleic acid-guided nuclease editingsystem may use two separate guide nucleic acid molecules that combine tofunction as a guide nucleic acid, e.g., a CRISPR RNA (crRNA) andtrans-activating CRISPR RNA (tracrRNA). In other aspects, the guidenucleic acid may be a single guide nucleic acid that includes both thecrRNA and tracrRNA sequences.

In general, a guide nucleic acid (e.g., gRNA) complexes with acompatible nucleic acid-guided nuclease and can then hybridize with atarget sequence, thereby directing the nuclease to the target sequence.A guide nucleic acid can be DNA or RNA; alternatively, a guide nucleicacid may comprise both DNA and RNA. In some embodiments, a guide nucleicacid may comprise modified or non-naturally occurring nucleotides. Incases where the guide nucleic acid comprises RNA, the gRNA may beencoded by a DNA sequence on a polynucleotide molecule such as aplasmid, linear construct, or the coding sequence may reside within anediting cassette. The sequence for the gRNA may be under the control ofa constitutive promoter, or, in some embodiments and preferably, aninducible promoter as described below.

A guide nucleic acid comprises a guide sequence, where the guidesequence is a polynucleotide sequence having sufficient complementaritywith a target sequence to hybridize with the target sequence and directsequence-specific binding of a complexed nucleic acid-guided nuclease tothe target sequence. The degree of complementarity between a guidesequence and the corresponding target sequence, when optimally alignedusing a suitable alignment algorithm, is about or more than about 50%,60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment maybe determined with the use of any suitable algorithm for aligningsequences. In some embodiments, a guide sequence is about or more thanabout 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length.In some embodiments, a guide sequence is less than about 75, 50, 45, 40,35, 30, 25, 20 nucleotides in length. Preferably the guide sequence is10-30 or 15-20 nucleotides long, or 15, 16, 17, 18, 19, or 20nucleotides in length.

In the present methods and compositions, the guide nucleic acid isprovided as a sequence to be expressed from a plasmid or vector andcomprises both the guide sequence and the scaffold sequence as a singletranscript under the control of a promoter, and in some embodiments, aninducible promoter. The guide nucleic acid can be engineered to target adesired target sequence by altering the guide sequence so that the guidesequence is complementary to a desired target sequence, thereby allowinghybridization between the guide sequence and the target sequence. Ingeneral, to generate an edit in the target sequence, the gRNA/nucleasecomplex binds to a target sequence as determined by the guide RNA, andthe nuclease recognizes a protospacer adjacent motif (PAM) sequenceadjacent to the target sequence. The target sequence can be anypolynucleotide endogenous or exogenous to a prokaryotic or eukaryoticcell, or in vitro. For example, the target sequence can be apolynucleotide residing in the nucleus of a eukaryotic cell. The targetsequence can be a sequence encoding a gene product (e.g., a protein) ora non-coding sequence (e.g., a regulatory polynucleotide, an intron, aPAM, or “junk” DNA).

The guide nucleic acid may be part of an editing cassette that encodesthe donor nucleic acid. Alternatively, the guide nucleic acid may not bepart of the editing cassette and instead may be encoded on the engine orediting vector backbone. For example, a sequence coding for a guidenucleic acid can be assembled or inserted into a vector backbone first,followed by insertion of the donor nucleic acid in, e.g., the editingcassette. In other cases, the donor nucleic acid in, e.g., an editingcassette can be inserted or assembled into a vector backbone first,followed by insertion of the sequence coding for the guide nucleic acid.In yet other cases, the sequence encoding the guide nucleic acid and thedonor nucleic acid (inserted, for example, in an editing cassette) aresimultaneously but separately inserted or assembled into a vector. Inyet other embodiments, the sequence encoding the guide nucleic acid andthe sequence encoding the donor nucleic acid are both included in theediting cassette.

The target sequence is associated with a proto-spacer mutation (PAM),which is a short nucleotide sequence recognized by the gRNA/nucleasecomplex. The precise preferred PAM sequence and length requirements fordifferent nucleic acid-guided nucleases vary; however, PAMs typicallyare 2-7 base-pair sequences adjacent or in proximity to the targetsequence and, depending on the nuclease, can be 5′ or 3′ to the targetsequence. Engineering of the PAM-interacting domain of a nucleicacid-guided nuclease may allow for alteration of PAM specificity,improve target site recognition fidelity, decrease target siterecognition fidelity, or increase the versatility of a nucleicacid-guided nuclease. In certain embodiments, the genome editing of atarget sequence both introduces a desired DNA change to a targetsequence, e.g., the genomic DNA of a cell, and removes, mutates, orrenders inactive a proto-spacer mutation (PAM) region in the targetsequence. Rendering the PAM at the target sequence inactive precludesadditional editing of the cell genome at that target sequence, e.g.,upon subsequent exposure to a nucleic acid-guided nuclease complexedwith a synthetic guide nucleic acid in later rounds of editing. Thus,cells having the desired target sequence edit and an altered PAM can beselected using a nucleic acid-guided nuclease complexed with a syntheticguide nucleic acid complementary to the target sequence. The genome ofthe cells that did not undergo the first editing event will be cutrendering a double-stranded DNA break, and thus these cells will notcontinue to be viable. The genome of the cells containing the desiredtarget sequence edit and PAM alteration will not be cut, as these editedcells no longer contain the necessary PAM site and will thus continue togrow and propagate.

The range of target sequences that nucleic acid-guided nucleases canrecognize is constrained by the need for a specific PAM to be locatednear the desired target sequence. As a result, it often can be difficultto target edits with the precision that is necessary for genome editing.It has been found that nucleases can recognize some PAMs very well(e.g., canonical PAMs), and other PAMs less well or poorly (e.g.,non-canonical PAMs). Because certain of the methods disclosed hereinallow for identification of edited cells in a background of uneditedcells (see, e.g., FIGS. 7A-7E and the descriptions thereof), the methodsallow for identification of edited cells where the PAM is less thanoptimal; that is, the methods for identifying edited cells herein allowfor identification of edited cells even if editing efficiency is verylow. Additionally, the present methods expand the scope of targetsequences that may be edited since edits are more readily identified,including cells where the genome edits are associated with lessfunctional PAMs.

As for the nuclease component of the nucleic acid-guided nucleaseediting system, a polynucleotide sequence encoding the nucleicacid-guided nuclease can be codon optimized for expression in particularcell types, such as archaeal, prokaryotic or eukaryotic cells.Eukaryotic cells can be yeast, fungi, algae, plant, animal, or humancells. Eukaryotic cells may be those of or derived from a particularorganism, such as a mammal, including but not limited to human, mouse,rat, rabbit, dog, or non-human mammals including non-human primates. Thechoice of nucleic acid-guided nuclease to be employed depends on manyfactors, such as what type of edit is to be made in the target sequenceand whether an appropriate PAM is located close to the desired targetsequence. Nucleases of use in the methods described herein include butare not limited to Cas 9, Cas 12/CpfI, MAD2, or MAD7 or other MADzymes.As with the guide nucleic acid, the nuclease may be encoded by a DNAsequence on a vector (e.g., the engine vector) and be under the controlof a constitutive or inducible promoter. In some embodiments, thesequence encoding the nuclease is under the control of an induciblepromoter, and the inducible promoter may be separate from but the sameas the inducible promoter controlling transcription of the guide nucleicacid; that is, a separate inducible promoter drives the transcription ofthe nuclease and guide nucleic acid sequences but the two induciblepromoters may be the same type of inducible promoter (e.g., both are pLpromoters). Alternatively, the inducible promoter controlling expressionof the nuclease may be different from the inducible promoter controllingtranscription of the guide nucleic acid; that is, e.g., the nuclease maybe under the control of the pBAD inducible promoter, and the guidenucleic acid may be under the control of the pL inducible promoter.

Another component of the nucleic acid-guided nuclease system is thedonor nucleic acid. In some embodiments, the donor nucleic acid is onthe same polynucleotide (e.g., editing vector or editing cassette) asthe guide nucleic acid and may be (but not necessarily) under thecontrol of the same promoter as the guide nucleic acid (e.g., a singlepromoter driving the transcription of both the guide nucleic acid andthe donor nucleic acid). The donor nucleic acid is designed to serve asa template for homologous recombination with a target sequence nicked orcleaved by the nucleic acid-guided nuclease as a part of thegRNA/nuclease complex. A donor nucleic acid polynucleotide may be of anysuitable length, such as about or more than about 20, 25, 50, 75, 100,150, 200, 500, or 1000 nucleotides in length. In certain preferredaspects, the donor nucleic acid can be provided as an oligonucleotide ofbetween 20-300 nucleotides, more preferably between 50-250 nucleotides.The donor nucleic acid comprises a region that is complementary to aportion of the target sequence (e.g., a homology arm). When optimallyaligned, the donor nucleic acid overlaps with (is complementary to) thetarget sequence by, e.g., about 20, 25, 30, 35, 40, 50, 60, 70, 80, 90or more nucleotides. In many embodiments, the donor nucleic acidcomprises two homology arms (regions complementary to the targetsequence) flanking the mutation or difference between the donor nucleicacid and the target template. The donor nucleic acid comprises at leastone mutation or alteration compared to the target sequence, such as aninsertion, deletion, modification, or any combination thereof comparedto the target sequence.

Often the donor nucleic acid is provided as an editing cassette, whichis inserted into a vector backbone where the vector backbone maycomprise a promoter driving transcription of the gRNA and the codingsequence of the gRNA, or the vector backbone may comprise a promoterdriving the transcription of the gRNA but not the gRNA itself. Moreover,there may be more than one, e.g., two, three, four, or more guidenucleic acid/donor nucleic acid cassettes inserted into an editingvector, where each guide nucleic acid is under the control of separatedifferent promoters, separate like promoters, or where all guide nucleicacid/donor nucleic acid pairs are under the control of a singlepromoter. In some embodiments, the promoter driving transcription of thegRNA and the donor nucleic acid (or driving more than one gRNA/donornucleic acid pair) is an inducible promoter and the promoter drivingtranscription of the nuclease is an inducible promoter as well. Foradditional information regarding editing cassettes, see U.S. Pat. Nos.9,982,278; 10,240,167; 10,266,849; 10,351,877; 10,364,442; and10,435,715; and U.S. Ser. Nos. 16/275,465 and 16/551,517.

In addition to the donor nucleic acid, an editing cassette may compriseone or more primer sites. The primer sites can be used to amplify theediting cassette by using oligonucleotide primers; for example, if theprimer sites flank one or more of the other components of the editingcassette.

Also, as described above, the donor nucleic acid may optionallycomprise—in addition to the at least one mutation relative to a targetsequence-one or more PAM sequence alterations that mutate, delete orrender inactive the PAM site in the target sequence. The PAM sequencealteration in the target sequence renders the PAM site “immune” to thenucleic acid-guided nuclease and protects the target sequence fromfurther editing in subsequent rounds of editing if the same nuclease isused.

In addition, the editing cassette may comprise a barcode. A barcode is aunique DNA sequence that corresponds to the donor DNA sequence such thatthe barcode can identify the edit made to the corresponding targetsequence. The barcode typically comprises four or more nucleotides. Insome embodiments, the editing cassettes comprise a collection of donornucleic acids representing, e.g., gene-wide or genome-wide libraries ofdonor nucleic acids. The library of editing cassettes is cloned intovector backbones where, e.g., each different donor nucleic acid isassociated with a different barcode.

Additionally, in some embodiments, an expression vector or cassetteencoding components of the nucleic acid-guided nuclease system furtherencodes a nucleic acid-guided nuclease comprising one or more nuclearlocalization sequences (NLSs), such as about or more than about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, the engineerednuclease comprises NLSs at or near the amino-terminus, NLSs at or nearthe carboxy-terminus, or a combination thereof.

The engine and editing vectors comprise control sequences operablylinked to the component sequences to be transcribed. As stated above,the promoters driving transcription of one or more components of thenucleic acid-guided nuclease editing system may be inducible such as oneor both of the gRNA and the nuclease. A number of gene regulationcontrol systems have been developed for the controlled expression ofgenes in plant, microbe, and animal cells, including mammalian cells,including the pL promoter (induced by heat inactivation of the CI857repressor), the pBAD promoter (induced by the addition of arabinose tothe cell growth medium), and the rhamnose inducible promoter (induced bythe addition of rhamnose to the cell growth medium). Other systemsinclude the tetracycline-controlled transcriptional activation system(Tet-On/Tet-Off, Clontech, Inc. (Palo Alto, Calif.); Bujard and Gossen,PNAS, 89(12):5547-5551 (1992)), the Lac Switch Inducible system(Wyborski et al., Environ Mol Mutagen, 28(4):447-58 (1996); DuCoeur etal., Strategies 5(3):70-72 (1992); U.S. Pat. No. 4,833,080), theecdysone-inducible gene expression system (No et al., PNAS,93(8):3346-3351 (1996)), the cumate gene-switch system (Mullick et al.,BMC Biotechnology, 6:43 (2006)), and the tamoxifen-inducible geneexpression (Zhang et al., Nucleic Acids Research, 24:543-548 (1996)) aswell as others. In the present methods used in the modules andinstruments described herein, it is preferred that at least one of thenucleic acid-guided nuclease editing components (e.g., the nucleaseand/or the gRNA) is under the control of a promoter that is activated bya rise in temperature, as such a promoter allows for the promoter to beactivated by an increase in temperature, and de-activated by a decreasein temperature, thereby “turning off” the editing process. Thus, in thescenario of a promoter that is de-activated by a decrease intemperature, editing in the cell can be turned off without having tochange media; to remove, e.g., an inducible biochemical in the mediumthat is used to induce editing.

Automated Multi-Module Cell Processing Instruments Comprising FTEPs

The present disclosure relates to flow-through electroporation devicesor modules that can be used alone, or as one module in automatedmulti-module cell processing instruments. In some embodiments, the FTEPmay be included as part of a reagent cartridge, wherein the reagentcartridge may further include sample receptacles, reagent receptacles,and/or waste receptacles, etc.; additionally, in certain embodiments,the reagent cartridge will comprise a script readable by a processor fordispensing the reagents and controlling the electroporation devicecontained within the reagent cartridge. An automated multi-module cellprocessing instrument with an FTEP can be used to process many differenttypes of cells in a controlled, contained, and reproducible manner,including bacterial cells, mammalian cells, non-mammalian eukaryoticcells, yeast cells, fungi, archaea, and the like.

Automated Cell Editing Instruments

FIG. 4A depicts an exemplary automated multi-module cell processinginstrument 400 to, e.g., perform one of the exemplary workflowscomprising a split protein reporter system as described herein. Theinstrument 400, for example, may be and preferably is designed as astand-alone desktop instrument for use within a laboratory environment.The instrument 400 may incorporate a mixture of reusable and disposablecomponents for performing the various integrated processes in conductingautomated genome cleavage and/or editing in cells without humanintervention. Illustrated is a gantry 402, providing an automatedmechanical motion system (actuator) (not shown) that supplies XYZ axismotion control to, e.g., an automated (i.e., robotic) liquid handlingsystem 458 including, e.g., an air displacement pipettor 432 whichallows for cell processing among multiple modules without humanintervention. In some automated multi-module cell processinginstruments, the air displacement pipettor 432 is moved by gantry 402and the various modules and reagent cartridges remain stationary;however, in other embodiments, the liquid handling system 458 may staystationary while the various modules and reagent cartridges are moved.Also included in the automated multi-module cell processing instrument400 are reagent cartridges 410 comprising reservoirs 412 andtransformation module 430 (e.g., a flow-through electroporation deviceas described in detail in relation to FIGS. 1A-1P), as well as washreservoirs 406, cell input reservoir 451 and cell output reservoir 453.The wash reservoirs 406 may be configured to accommodate large tubes,for example, wash solutions, or solutions that are used often throughoutan iterative process. Although two of the reagent cartridges 410comprise a wash reservoir 406 in FIG. 4A, the wash reservoirs insteadcould be included in a wash cartridge where the reagent and washcartridges are separate cartridges. In such a case, the reagentcartridge 410 and wash cartridge 404 may be identical except for theconsumables (reagents or other components contained within the variousinserts) inserted therein. (See, e.g., FIGS. 2A and 2B.)

In some implementations, the reagent cartridges 410 are disposable kitscomprising reagents and cells for use in the automated multi-module cellprocessing/editing instrument 400. For example, a user may open andposition each of the reagent cartridges 410 comprising various desiredinserts and reagents within the chassis of the automated multi-modulecell editing instrument 400 prior to activating cell processing.Further, each of the reagent cartridges 410 may be inserted intoreceptacles in the chassis having different temperature zonesappropriate for the reagents contained therein.

Also illustrated in FIG. 4A is the robotic liquid handling system 458including the gantry 402 and air displacement pipettor 432. In someexamples, the robotic handling system 458 may include an automatedliquid handling system such as those manufactured by Tecan Group Ltd. ofMannedorf, Switzerland, Hamilton Company of Reno, Nev. (see, e.g.,WO2018015544A1), or Beckman Coulter, Inc. of Fort Collins, Colo. (see,e.g., US20160018427A1). Pipette tips may be provided in a pipettetransfer tip supply (not shown) for use with the air displacementpipettor 432.

Inserts or components of the reagent cartridges 410, in someimplementations, are marked with machine-readable indicia (not shown),such as bar codes, for recognition by the robotic handling system 458.For example, the robotic liquid handling system 458 may scan one or moreinserts within each of the reagent cartridges 410 to confirm contents.In other implementations, machine-readable indicia may be marked uponeach reagent cartridge 410, and a processing system (not shown, but seeelement 437 of FIG. 4B) of the automated multi-module cell editinginstrument 400 may identify a stored materials map based upon themachine-readable indicia. In the embodiment illustrated in FIG. 4A, acell growth module comprises a cell growth vial 418 (described ingreater detail below in relation to FIGS. 5A-5D). Additionally seen isthe TFF module 422 (described above in detail in relation to FIGS.6A-6E) and selection module 420. Also illustrated as part of theautomated multi-module cell processing instrument 400 of FIG. 4A is asingulation module 440 (e.g., a solid wall isolation, incubation andnormalization device (SWIIN device) is shown here) described herein inrelation to FIGS. 7A-7E, served by, e.g., robotic liquid handing system458 and air displacement pipettor 432. Additionally seen is a selectionmodule 420. Also note the placement of three heatsinks 455.

FIG. 4B is a simplified representation of the contents of the exemplarymulti-module cell processing instrument 400 depicted in FIG. 4A.Cartridge-based source materials (such as in reagent cartridges 410),for example, may be positioned in designated areas on a deck of theinstrument 400 for access by an air displacement pipettor 432. The deckof the multi-module cell processing instrument 400 may include aprotection sink such that contaminants spilling, dripping, oroverflowing from any of the modules of the instrument 400 are containedwithin a lip of the protection sink. Also seen are reagent cartridges410, which are shown disposed with thermal assemblies 411 which cancreate temperature zones appropriate for different regions. Note thatone of the reagent cartridges also comprises a flow-throughelectroporation device 430 (FTEP), served by FTEP interface (e.g.,manifold arm) and actuator 431. Also seen is TFF module 422 withadjacent thermal assembly 425, where the TFF module is served by TFFinterface (e.g., manifold arm) and actuator 433. Thermal assemblies 425,435, and 445 encompass thermal electric devices such as Peltier devices,as well as heatsinks, fans and coolers. The rotating growth vial 418 iswithin a growth module 434, where the growth module is served by twothermal assemblies 435. A selection module is seen at 420. Also seen isthe SWIIN module 440, comprising a SWIIN cartridge 441, where the SWIINmodule also comprises a thermal assembly 445, illumination 443 (in thisembodiment, backlighting), evaporation and condensation control 449, andwhere the SWIIN module is served by SWIIN interface (e.g., manifold arm)and actuator 447. Also seen in this view is touch screen display 401,display actuator 403, illumination 405 (one on either side ofmulti-module cell processing instrument 400), and cameras 439 (oneillumination device on either side of multi-module cell processinginstrument 400). Finally, element 437 comprises electronics, such ascircuit control boards, high-voltage amplifiers, power supplies, andpower entry; as well as pneumatics, such as pumps, valves and sensors.

FIG. 4C illustrates a front perspective view of multi-module cellprocessing instrument 400 for use in as a desktop version of theautomated multi-module cell editing instrument 400. For example, achassis 490 may have a width of about 24-48 inches, a height of about24-48 inches and a depth of about 24-48 inches. Chassis 490 may be andpreferably is designed to hold all modules and disposable supplies usedin automated cell processing and to perform all processes requiredwithout human intervention; that is, chassis 490 is configured toprovide an integrated, stand-alone automated multi-module cellprocessing instrument. As illustrated in FIG. 4C, chassis 490 includestouch screen display 401, cooling grate 464, which allows for air flowvia an internal fan (not shown). The touch screen display providesinformation to a user regarding the processing status of the automatedmulti-module cell editing instrument 400 and accepts inputs from theuser for conducting the cell processing. In this embodiment, the chassis490 is lifted by adjustable feet 470 a, 470 b, 470 c and 470 d (feet 470a-470 c are shown in this FIG. 4C). Adjustable feet 470 a-470 d, forexample, allow for additional air flow beneath the chassis 490.

Inside the chassis 490, in some implementations, will be most or all ofthe components described in relation to FIGS. 4A and 4B, including therobotic liquid handling system disposed along a gantry, reagentcartridges 410 including a flow-through electroporation device, arotating growth vial 418 in a cell growth module 434, a tangential flowfiltration module 422, a SWIIN module 440 as well as interfaces andactuators for the various modules. In addition, chassis 490 housescontrol circuitry, liquid handling tubes, air pump controls, valves,sensors, thermal assemblies (e.g., heating and cooling units) and othercontrol mechanisms. For examples of multi-module cell editinginstruments, see U.S. Pat. No. 10,253,316, issued 9 Apr. 2019; U.S. Pat.No. 10,329,559, issued 25 Jun. 2019; U.S. Pat. No. 10,323,242, issued 18Jun. 2019; U.S. Pat. No. 10,421,959, issued 24 Sep. 2019; U.S. Pat. No.10,465,185, issued 5 Nov. 2019; and U.S. Pat. No. 10,519,437, issued 31Dec. 2019; and U.S. Ser. No. 16/666,964, filed 29 Oct. 2019; and Ser.No. 16/680,643, filed 12 Nov. 2019 all of which are herein incorporatedby reference in their entirety.

Rotating Cell Growth Module

FIG. 5A shows one embodiment of a rotating growth vial 500 for use withthe cell growth device described herein. The rotating growth vial is anoptically-transparent container having an open end 504 for receivingliquid media and cells, a central vial region 506 that defines theprimary container for growing cells, a tapered-to-constricted region 518defining at least one light path 510, a closed end 516, and a driveengagement mechanism 512. The rotating growth vial has a centrallongitudinal axis 520 around which the vial rotates, and the light path510 is generally perpendicular to the longitudinal axis of the vial. Thefirst light path 510 is positioned in the lower constricted portion ofthe tapered-to-constricted region 518. Optionally, some embodiments ofthe rotating growth vial 500 have a second light path 508 in the taperedregion of the tapered-to-constricted region 518. Both light paths inthis embodiment are positioned in a region of the rotating growth vialthat is constantly filled with the cell culture (cells+growth media) andis not affected by the rotational speed of the growth vial. The firstlight path 510 is shorter than the second light path 508 allowing forsensitive measurement of OD values when the OD values of the cellculture in the vial are at a high level (e.g., later in the cell growthprocess), whereas the second light path 508 allows for sensitivemeasurement of OD values when the OD values of the cell culture in thevial are at a lower level (e.g., earlier in the cell growth process).Also shown is lip 502, which allows the rotating growth vial to beseated in a growth module (not shown) and further allows for easyhandling by the user.

In some configurations of the rotating growth vial, the rotating growthvial has two or more “paddles” or interior features disposed within therotating growth vial, extending from the inner wall of the rotatinggrowth vial toward the center of the central vial region 506. In someaspects, the width of the paddles or features varies with the size orvolume of the rotating growth vial, and may range from 1/20 to just over⅓ the diameter of the rotating growth vial, or from 1/15 to ¼ thediameter of the rotating growth vial, or from 1/10 to ⅕ the diameter ofthe rotating growth vial. In some aspects, the length of the paddlesvaries with the size or volume of the rotating growth vial, and mayrange from ⅘ to ¼ the length of the main body of the rotating growthvial 500, or from ¾ to ⅓ the length of the central body region 506 ofthe rotating growth vial, or from ½ to ⅓ the length of the central bodyregion 506 of the rotating growth vial 500. In other aspects, there maybe concentric rows of raised features disposed on the inner surface ofthe main body of the rotating growth vial arranged horizontally orvertically; and in other aspects, there may be a spiral configuration ofraised features disposed on the inner surface of the main body of therotating growth vial. In alternative aspects, the concentric rows ofraised features or spiral configuration may be disposed upon a post orcenter structure of the rotating growth vial. Though described above ashaving two paddles, the rotating growth vial 500 may comprise 3, 4, 5, 6or more paddles, and up to 20 paddles. The number of paddles will dependupon, e.g., the size or volume of the rotating growth vial 500. Thepaddles may be arranged symmetrically as single paddles extending fromthe inner wall of the vial into the interior of the vial, or the paddlesmay be symmetrically arranged in groups of 2, 3, 4 or more paddles in agroup (for example, a pair of paddles opposite another pair of paddles)extending from the inner wall of the vial into the interior of the vial.In another embodiment, the paddles may extend from the middle of therotating growth vial out toward the wall of the rotating growth vial,from, e.g., a post or other support structure in the interior of therotating growth vial.

The drive engagement mechanism 512 engages with a motor (not shown) torotate the vial. In some embodiments, the motor drives the driveengagement mechanism 512 such that the rotating growth vial is rotatedin one direction only, and in other embodiments, the rotating growthvial is rotated in a first direction for a first amount of time orperiodicity, rotated in a second direction (i.e., the oppositedirection) for a second amount of time or periodicity, and this processmay be repeated so that the rotating growth vial (and the cell culturecontents) are subjected to an oscillating motion. Further, the choice ofwhether the culture is subject to oscillation and the periodicitytherefor may be selected by the user. The first amount of time and thesecond amount of time may be the same or may be different. The amount oftime may be 1, 2, 3, 4, 5, or more seconds, or may be 1, 2, 3, 4 or moreminutes. In another embodiment, in an early stage of cell growth, therotating growth vial may be oscillated at a first periodicity (e.g.,every 60 seconds), and then at a later stage of cell growth, therotating growth vial may be oscillated at a second periodicity (e.g.,every one second) different from the first periodicity.

The rotating growth vial 500 may be reusable or, preferably, therotating growth vial is consumable. In some embodiments, the rotatinggrowth vial is consumable and is presented to the user pre-filled withgrowth medium, where the vial is hermetically sealed at the open end 504with a foil or film seal. A medium-filled rotating growth vial packagedin such a manner may be part of a kit for use with a stand-alone cellgrowth device or with a cell growth module that is part of an automatedmulti-module cell processing instrument. To introduce cells into thevial, a user need only pipette up a desired volume of cells and use thepipette tip to punch through the foil or film seal of the vial. Open end504 may optionally include an extended lip 502 to overlap and engagewith the cell growth device (not shown). In automated systems, therotating growth vial 500 may be tagged with a barcode or otheridentifying means that can be read by a scanner or camera that is partof the automated instrument (not shown).

The volume of the rotating growth vial 500 and the volume of the cellculture (including growth medium) may vary greatly, but the volume ofthe rotating growth vial 500 must be large enough for the cell culturein the growth vial to get proper aeration while the vial is rotating andto generate an adequate number of cells. In practice, the volume of therotating growth vial 500 may range from 1-250 ml, 2-100 ml, from 5-80ml, 10-50 ml, or from 12-35 ml. Likewise, the volume of the cell culture(cells+growth media) should be appropriate to allow proper aeration inthe rotating growth vial. Thus, the volume of the cell culture should beapproximately 5-85% of the volume of the growth vial or from 20-60% ofthe volume of the growth vial. For example, for a 35 ml growth vial, thevolume of the cell culture would be from about 1.8 ml to about 27 ml, orfrom 5 ml to about 21 ml.

The rotating growth vial 500 preferably is fabricated from abio-compatible optically transparent material or at least the portion ofthe vial comprising the light path(s) is transparent. Additionally,material from which the rotating growth vial is fabricated should beable to be cooled to about 4° C. or lower and heated to about 55° C. orhigher to accommodate both temperature-based cell assays and long-termstorage at low temperatures. Further, the material that is used tofabricate the vial must be able to withstand temperatures up to 55° C.without deformation while spinning. Suitable materials include glass,cyclic olefin copolymer (COC), polyvinyl chloride, polyethylene,polyamide, polypropylene, polycarbonate, poly(methyl methacrylate(PMMA), polysulfone, polyurethane, and co-polymers of these and otherpolymers. Preferred materials include polypropylene, polycarbonate, orpolystyrene. In some embodiments, the rotating growth vial isinexpensively fabricated by, e.g., injection molding or extrusion.

FIGS. 5B-5D show an embodiment of a cell growth module 550 comprising arotating growth vial 500. FIG. 5B is a perspective view of oneembodiment of a cell growth module 550. FIG. 5C depicts a cut-away viewof the cell growth module 550 from FIG. 5B. In both figures, therotating growth vial 500 is seen positioned inside a main housing 526with the extended lip 502 of the rotating growth vial 500 extendingabove the main housing 526. Additionally, end housings 522, a lowerhousing 532, and flanges 524 are indicated in both figures. Flanges 524are used to attach the cell growth device/module to heating/coolingmeans or to another structure (not shown). FIG. 5C depicts additionaldetail. In FIG. 5C, upper bearing 542 and lower bearing 530 are shownpositioned in main housing 526. Upper bearing 542 and lower bearing 530support the vertical load of rotating growth vial 500. Lower housing 532contains the drive motor 536. The cell growth device 550 of FIG. 5Ccomprises two light paths: a primary light path 534, and a secondarylight path 530. Light path 534 corresponds to light path 510 positionedin the constricted portion of the tapered-to-constricted portion of therotating growth vial, and light path 530 corresponds to light path 508in the tapered portion of the tapered-to-constricted portion of therotating growth vial. Light paths 510 and 508 are not shown in FIG. 5Cbut may be seen in, e.g., FIG. 5A. In addition to light paths 534 and530, there is an emission board 528 to illuminate the light path(s), anddetector board 546 to detect the light after the light travels throughthe cell culture liquid in the rotating growth vial 500.

The drive motor 536 used to rotate the rotating growth vial 500 in someembodiments is a brushless DC type drive motor with built-in drivecontrols that can be set to hold a constant revolution per minute (RPM)between 0 and about 3000 RPM. Alternatively, other motor types such as astepper, servo, brushed DC, and the like can be used. Optionally, thedrive motor 506 may also have direction control to allow reversing ofthe rotational direction, and a tachometer to sense and report actualRPM. The motor is controlled by a processor (not shown) according to,e.g., standard protocols programmed into the processor and/or userinput, and the motor may be configured to vary RPM to cause axialprecession of the cell culture thereby enhancing mixing, e.g., toprevent cell aggregation, increase aeration, and optimize cellularrespiration.

Main housing 526, end housings 522 and lower housing 532 of the cellgrowth device/module 550 may be fabricated from any suitable, robustmaterial including aluminum, stainless steel, and other thermallyconductive materials, including plastics. These structures or portionsthereof can be created through various techniques, e.g., metalfabrication, injection molding, creation of structural layers that arefused, etc. Whereas the rotating growth vial 500 is envisioned in someembodiments to be reusable but preferably is consumable, the othercomponents of the cell growth device 550 are preferably reusable and canfunction as a stand-alone benchtop device or, as here, as a module in amulti-module cell processing instrument.

The processor (not shown) of the cell growth system may be programmedwith information to be used as a “blank” or control for the growing cellculture. A “blank” or control is a vessel containing cell growth mediumonly, which yields 100% transmittance and 0 OD, while the cell samplewill deflect light rays and will have a lower percent transmittance andhigher OD. As the cells grow in the media and become denser,transmittance will decrease and OD will increase. The processor of thecell growth system may be programmed to use wavelength values for blankscommensurate with the growth media typically used in cell culture(whether, e.g., mammalian cells, bacterial cells, animal cells, yeastcells, etc.). Alternatively, a second spectrophotometer and vessel maybe included in the cell growth system, where the secondspectrophotometer is used to read a blank at designated intervals.

FIG. 5D illustrates a cell growth device/module 550 as part of anassembly comprising the cell growth device 550 of FIG. 5B coupled tolight source 590, detector 592, and thermoelectric components 594. Therotating growth vial 500 is inserted into the cell growth device 550.Components of the light source 590 and detector 592 (e.g., such as aphotodiode with gain control to cover 5-log) are coupled to the mainhousing of the cell growth device 550. The lower housing 532 that housesthe motor that rotates the rotating growth vial is illustrated, as isone of the flanges 524 that secures the cell growth device to theassembly. Also illustrated is a Peltier device or thermoelectriccomponent 594. In this embodiment, thermal control is accomplished byattachment and electrical integration of the cell growth device 500 tothe thermoelectric component 594 via the flange 504 on the base of thelower housing 532. Thermoelectric coolers/devices 594 are capable of“pumping” heat to either side of a junction, either cooling a surface orheating a surface depending on the direction of current flow. In oneembodiment, a thermistor is used to measure the temperature of the mainhousing and then, through a standard electronicproportional-integral-derivative (PID) controller loop, the rotatinggrowth vial 500 is controlled to approximately +/−0.5° C.

In certain embodiments, a rear-mounted power entry module contains thesafety fuses and the on-off switch, which when switched on powers theinternal AC and DC power supplies (not shown) activating the processor.Measurements of optical densities (OD) at programmed time intervals areaccomplished using a 600 nm Light Emitting Diode (LED) (not shown) thathas been columnated through an optic into the lower constricted portionof the rotating growth vial which contains the cells of interest. Thelight continues through a collection optic to the detection system whichconsists of a (digital) gain-controlled silicone photodiode. Generally,optical density is normally shown as the absolute value of the logarithmwith base 10 of the power transmission factors of an optical attenuator:OD=−log 10 (Power out/Power in). Since OD is the measure of opticalattenuation—that is, the sum of absorption, scattering, andreflection—the cell growth device OD measurement records the overallpower transmission, so as the cells grow and become denser inpopulation, the OD (the loss of signal) increases. The OD system ispre-calibrated against OD standards with these values stored in anon-board memory accessible by the measurement program.

In use, cells are inoculated (cells can be pipetted, e.g., from anautomated liquid handling system or by a user) into pre-filled growthmedia of a rotating growth vial 500 by piercing though the foil or filmseal. The programmed software of the cell growth device 550 sets thecontrol temperature for growth, typically 30° C., then slowly starts therotation of the rotating growth vial. The cell/growth media mixtureslowly moves vertically up the wall due to centrifugal force allowingthe rotating growth vial to expose a large surface area of the mixtureto a normal oxygen environment. The growth monitoring system takeseither continuous readings of the OD or OD measurements at pre-set orpre-programmed time intervals. These measurements are stored in internalmemory and if requested the software plots the measurements versus timeto display a growth curve. If enhanced mixing is required, e.g., tooptimize growth conditions, the speed of the vial rotation can be variedto cause an axial precession of the liquid, and/or a completedirectional change can be performed at programmed intervals. The growthmonitoring can be programmed to automatically terminate the growth stageat a pre-determined OD, and then quickly cool the mixture to a lowertemperature to inhibit further growth.

One application for the cell growth device 550 is to constantly measurethe optical density of a growing cell culture. One advantage of thedescribed cell growth device is that optical density can be measuredcontinuously (kinetic monitoring) or at specific time intervals; e.g.,every 5, 10, 15, 20, 30 45, or 60 seconds, or every 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 minutes. While the cell growth device has been described inthe context of measuring the optical density (OD) of a growing cellculture, it should, however, be understood by a skilled artisan giventhe teachings of the present specification that other cell growthparameters can be measured in addition to or instead of cell culture OD.For example, spectroscopy using visible, UV, or near infrared (NIR)light allows monitoring the concentration of nutrients and/or wastes inthe cell culture. Additionally, spectroscopic measurements may be usedto quantify multiple chemical species simultaneously. Nonsymmetricchemical species may be quantified by identification of characteristicabsorbance features in the NIR. Conversely, symmetric chemical speciescan be readily quantified using Raman spectroscopy. Many criticalmetabolites, such as glucose, glutamine, ammonia, and lactate havedistinct spectral features in the IR, such that they may be easilyquantified. The amount and frequencies of light absorbed by the samplecan be correlated to the type and concentration of chemical speciespresent in the sample. Each of these measurement types provides specificadvantages. FT-NIR provides the greatest light penetration depth and canbe used for thicker samples. FT-mid-IR (MIR) provides information thatis more easily discernible as being specific for certain analytes asthese wavelengths are closer to the fundamental IR absorptions. FT-Ramanis advantageous when interference due to water is to be minimized. Otherspectral properties can be measured via, e.g., dielectric impedencespectroscopy, visible fluorescence, fluorescence polarization, orluminescence. Additionally, the cell growth device may includeadditional sensors for measuring, e.g., dissolved oxygen, carbondioxide, pH, conductivity, and the like.

Cell Concentration Module

As described above in relation to the rotating growth vial and cellgrowth module, in order to obtain an adequate number of cells fortransformation or transfection, cells typically are grown to a specificoptical density in medium appropriate for the growth of the cells ofinterest; however, for effective transformation or transfection, it isdesirable to decrease the volume of the cells as well as render thecells competent via buffer or medium exchange. Thus, one sub-componentor module that is desired in cell processing systems for the processeslisted above is a module or component that can grow, perform bufferexchange, and/or concentrate cells and render them competent so thatthey may be transformed or transfected with the nucleic acids needed forengineering or editing the cell's genome.

FIG. 6A shows a retentate member 622 (top), permeate member 620 (middle)and a tangential flow assembly 610 (bottom) comprising the retentatemember 622, membrane 624 (not seen in FIG. 6A), and permeate member 620(also not seen). In FIG. 6A, retentate member 622 comprises a tangentialflow channel 602, which has a serpentine configuration that initiates atone lower corner of retentate member 622 specifically at retentate port628—traverses across and up then down and across retentate member 622,ending in the other lower corner of retentate member 622 at a secondretentate port 628. Also seen on retentate member 622 are energydirectors 691, which circumscribe the region where a membrane or filter(not seen in this FIG. 6A) is seated, as well as interdigitate betweenareas of channel 602. Energy directors 691 in this embodiment mate withand serve to facilitate ultrasonic welding or bonding of retentatemember 622 with permeate/filtrate member 620 via the energy directorcomponent 691 on permeate/filtrate member 620 (at right). Additionally,countersinks 623 can be seen, two on the bottom one at the top middle ofretentate member 622. Countersinks 623 are used to couple and tangentialflow assembly 610 to a reservoir assembly (not seen in this FIG. 6A butsee FIG. 6B).

Permeate/filtrate member 620 is seen in the middle of FIG. 6A andcomprises, in addition to energy director 691, through-holes forretentate ports 628 at each bottom corner (which mate with thethrough-holes for retentate ports 628 at the bottom corners of retentatemember 622), as well as a tangential flow channel 602 and twopermeate/filtrate ports 626 positioned at the top and center of permeatemember 620. The tangential flow channel 602 structure in this embodimenthas a serpentine configuration and an undulating geometry, althoughother geometries may be used. Permeate member 620 also comprisescountersinks 623, coincident with the countersinks 623 on retentatemember 620.

At bottom of FIG. 6A is a tangential flow assembly 610 comprising theretentate member 622 positioned on top of an assembled with permeatemember 620. In this view, retentate member 622 is “on top” of the view,a membrane (not seen in this view of the assembly) would be adjacent andunder retentate member 622 and permeate member 620 (also not seen inthis view of the assembly) is adjacent to and beneath the membrane.Again countersinks 623 are seen, where the countersinks in the retentatemember 622 and the permeate member 620 are coincident and configured tomate with threads or mating elements for the countersinks disposed on areservoir assembly (not seen in FIG. 6A but see FIG. 6B).

A membrane or filter is disposed between the retentate and permeatemembers, where fluids can flow through the membrane but cells cannot andare thus retained in the flow channel disposed in the retentate member.Filters or membranes appropriate for use in the TFF device/module arethose that are solvent resistant, are contamination free duringfiltration, and are able to retain the types and sizes of cells ofinterest. For example, in order to retain small cell types such asbacterial cells, pore sizes can be as low as 0.2 μm, however for othercell types, the pore sizes can be as high as 20 m. Indeed, the poresizes useful in the TFF device/module include filters with sizes from0.20 μm, 0.21 μm, 0.22 μm, 0.23 μm, 0.24 μm, 0.25 μm, 0.26 μm, 0.27 μm,0.28 m, 0.29 μm, 0.30 μm, 0.31 μm, 0.32 μm, 0.33 μm, 0.34 μm, 0.35 μm,0.36 μm, 0.37 μm, 0.38 μm, 0.39 μm, 0.40 μm, 0.41 μm, 0.42 μm, 0.43 μm,0.44 μm, 0.45 μm, 0.46 μm, 0.47 μm, 0.48 μm, 0.49 μm, 0.50 μm andlarger. The filters may be fabricated from any suitable non-reactivematerial including cellulose mixed ester (cellulose nitrate and acetate)(CME), polycarbonate (PC), polyvinylidene fluoride (PVDF),polyethersulfone (PES), polytetrafluoroethylene (PTFE), nylon, glassfiber, or metal substrates as in the case of laser or electrochemicaletching.

The length of the channel structure 602 may vary depending on the volumeof the cell culture to be grown and the optical density of the cellculture to be concentrated. The length of the channel structuretypically is from 60 mm to 300 mm, or from 70 mm to 200 mm, or from 80mm to 100 mm. The cross-section configuration of the flow channel 402may be round, elliptical, oval, square, rectangular, trapezoidal, orirregular. If square, rectangular, or another shape with generallystraight sides, the cross section may be from about 10 μm to 1000 μmwide, or from 200 μm to 800 μm wide, or from 300 μm to 700 μm wide, orfrom 400 μm to 600 μm wide; and from about 10 μm to 1000 m high, or from200 μm to 800 μm high, or from 300 μm to 700 μm high, or from 400 m to600 μm high. If the cross section of the flow channel 102 is generallyround, oval or elliptical, the radius of the channel may be from about50 μm to 1000 μm in hydraulic radius, or from 5 μm to 800 μm inhydraulic radius, or from 200 μm to 700 μm in hydraulic radius, or from300 μm to 600 μm wide in hydraulic radius, or from about 200 to 500 μmin hydraulic radius. Moreover, the volume of the channel in theretentate 422 and permeate 620 members may be different depending on thedepth of the channel in each member.

FIG. 6B shows front perspective (upper figure) and rear perspective(lower figure) views of a reservoir assembly 650 configured to be usedwith the tangential flow assembly 610 seen in FIG. 6A. Seen in the frontperspective view (e.g., “front” being the side of reservoir assembly 650that is coupled to the tangential flow assembly 610 seen in FIG. 6A) areretentate reservoirs 652 on either side of permeate reservoir 654. Alsoseen are permeate ports 626, retentate ports 628, and three threads ormating elements 625 for countersinks 623 (countersinks 623 not seen inthis FIG. 6B). Threads or mating elements 625 for countersinks 623 areconfigured to mate or couple the tangential flow assembly 610 (seen inFIG. 6A) to reservoir assembly 650. Alternatively or in addition,fasteners, sonic welding or heat stakes may be used to mate or couplethe tangential flow assembly 610 to reservoir assembly 650. In additionis seen gasket 645 covering the top of reservoir assembly 650. Gasket645 is described in detail in relation to FIG. 6E. At left in FIG. 6B isa rear perspective view of reservoir assembly 650, where “rear” is theside of reservoir assembly 650 that is not coupled to the tangentialflow assembly. Seen are retentate reservoirs 652, permeate reservoir654, and gasket 645.

The TFF device may be fabricated from any robust material in whichchannels (and channel branches) may be milled including stainless steel,silicon, glass, aluminum, or plastics including cyclic-olefin copolymer(COC), cyclo-olefin polymer (COP), polystyrene, polyvinyl chloride,polyethylene, polyamide, polyethylene, polypropylene, acrylonitrilebutadiene, polycarbonate, polyetheretheketone (PEEK), poly(methylmethylacrylate) (PMMA), polysulfone, and polyurethane, and co-polymersof these and other polymers. If the TFF device/module is disposable,preferably it is made of plastic. In some embodiments, the material usedto fabricate the TFF device/module is thermally-conductive so that thecell culture may be heated or cooled to a desired temperature. Incertain embodiments, the TFF device is formed by precision mechanicalmachining, laser machining, electro discharge machining (for metaldevices); wet or dry etching (for silicon devices); dry or wet etching,powder or sandblasting, photostructuring (for glass devices); orthermoforming, injection molding, hot embossing, or laser machining (forplastic devices) using the materials mentioned above that are amenableto this mass production techniques.

FIG. 6C depicts a top-down view of the reservoir assemblies 650 shown inFIG. 6B. FIG. 6D depicts a cover 644 for reservoir assembly 650 shown inFIGS. 6B and 6E depicts a gasket 645 that in operation is disposed oncover 644 of reservoir assemblies 650 shown in FIG. 6B. FIG. 6C is atop-down view of reservoir assembly 650, showing the tops of the tworetentate reservoirs 652, one on either side of permeate reservoir 654.Also seen are grooves 632 that will mate with a pneumatic port (notshown), and fluid channels 634 that reside at the bottom of retentatereservoirs 652, which fluidically couple the retentate reservoirs 652with the retentate ports 628 (not shown), via the through-holes for theretentate ports in permeate member 620 and membrane 624 (also notshown). FIG. 6D depicts a cover 644 that is configured to be disposedupon the top of reservoir assembly 650. Cover 644 has round cut-outs atthe top of retentate reservoirs 652 and permeate/filtrate reservoir 654.Again at the bottom of retentate reservoirs 652 fluid channels 634 canbe seen, where fluid channels 634 fluidically couple retentatereservoirs 652 with the retentate ports 628 (not shown). Also shown arethree pneumatic ports 630 for each retentate reservoir 652 andpermeate/filtrate reservoir 654. FIG. 6E depicts a gasket 645 that isconfigures to be disposed upon the cover 644 of reservoir assembly 650.Seen are three fluid transfer ports 642 for each retentate reservoir 652and for permeate/filtrate reservoir 654. Again, three pneumatic ports630, for each retentate reservoir 652 and for permeate/filtratereservoir 654, are shown.

The overall work flow for cell growth comprises loading a cell cultureto be grown into a first retentate reservoir, optionally bubbling air oran appropriate gas through the cell culture, passing or flowing the cellculture through the first retentate port then tangentially through theTFF channel structure while collecting medium or buffer through one orboth of the permeate ports 606, collecting the cell culture through asecond retentate port 604 into a second retentate reservoir, optionallyadding additional or different medium to the cell culture and optionallybubbling air or gas through the cell culture, then repeating theprocess, all while measuring, e.g., the optical density of the cellculture in the retentate reservoirs continuously or at desiredintervals. Measurements of optical densities (OD) at programmed timeintervals are accomplished using a 600 nm Light Emitting Diode (LED)that has been columnated through an optic into the retentatereservoir(s) containing the growing cells. The light continues through acollection optic to the detection system which consists of a (digital)gain-controlled silicone photodiode. Generally, optical density is shownas the absolute value of the logarithm with base 10 of the powertransmission factors of an optical attenuator: OD=−log 10 (Powerout/Power in). Since OD is the measure of optical attenuation—that is,the sum of absorption, scattering, and reflection—the TFF device ODmeasurement records the overall power transmission, so as the cells growand become denser in population, the OD (the loss of signal) increases.The OD system is pre-calibrated against OD standards with these valuesstored in an on-board memory accessible by the measurement program.

In the channel structure, the membrane bifurcating the flow channelsretains the cells on one side of the membrane (the retentate side 622)and allows unwanted medium or buffer to flow across the membrane into afiltrate or permeate side (e.g., permeate member 620) of the device.Bubbling air or other appropriate gas through the cell culture bothaerates and mixes the culture to enhance cell growth. During theprocess, medium that is removed during the flow through the channelstructure is removed through the permeate/filtrate ports 606.Alternatively, cells can be grown in one reservoir with bubbling oragitation without passing the cells through the TFF channel from onereservoir to the other.

The overall workflow for cell concentration using the TFF device/moduleinvolves flowing a cell culture or cell sample tangentially through thechannel structure. As with the cell growth process, the membranebifurcating the flow channels retains the cells on one side of themembrane and allows unwanted medium or buffer to flow across themembrane into a permeate/filtrate side (e.g., permeate member 620) ofthe device. In this process, a fixed volume of cells in medium or bufferis driven through the device until the cell sample is collected into oneof the retentate ports 604, and the medium/buffer that has passedthrough the membrane is collected through one or both of thepermeate/filtrate ports 606. All types of prokaryotic and eukaryoticcells-both adherent and non-adherent cells—can be grown in the TFFdevice. Adherent cells may be grown on beads or other cell scaffoldssuspended in medium that flow through the TFF device.

The medium or buffer used to suspend the cells in the cell concentrationdevice/module may be any suitable medium or buffer for the type of cellsbeing transformed or transfected, such as LB, SOC, TPD, YPG, YPAD, MEM,DMEM, IMDM, RPMI, Hanks', PBS and Ringer's solution, where the media maybe provided in a reagent cartridge as part of a kit. For culture ofadherent cells, cells may be disposed on beads, microcarriers, or othertype of scaffold suspended in medium. Most normal mammaliantissue-derived cells—except those derived from the hematopoieticsystem—are anchorage dependent and need a surface or cell culturesupport for normal proliferation. In the rotating growth vial describedherein, microcarrier technology is leveraged. Microcarriers ofparticular use typically have a diameter of 100-300 μm and have adensity slightly greater than that of the culture medium (thusfacilitating an easy separation of cells and medium for, e.g., mediumexchange) yet the density must also be sufficiently low to allowcomplete suspension of the carriers at a minimum stirring rate in orderto avoid hydrodynamic damage to the cells. Many different types ofmicrocarriers are available, and different microcarriers are optimizedfor different types of cells. There are positively charged carriers,such as Cytodex 1 (dextran-based, GE Healthcare), DE-52(cellulose-based, Sigma-Aldrich Labware), DE-53 (cellulose-based,Sigma-Aldrich Labware), and HLX 11-170 (polystyrene-based); collagen- orECM—(extracellular matrix) coated carriers, such as Cytodex 3(dextran-based, GE Healthcare) or HyQ-sphere Pro-F 102-4(polystyrene-based, Thermo Scientific); non-charged carriers, likeHyQ-sphere P 102-4 (Thermo Scientific); or macroporous carriers based ongelatin (Cultisphere, Percell Biolytica) or cellulose (Cytopore, GEHealthcare).

In both the cell growth and concentration processes, passing the cellsample through the TFF device and collecting the cells in one of theretentate ports 604 while collecting the medium in one of thepermeate/filtrate ports 606 is considered “one pass” of the cell sample.The transfer between retentate reservoirs “flips” the culture. Theretentate and permeatee ports collecting the cells and medium,respectively, for a given pass reside on the same end of TFFdevice/module with fluidic connections arranged so that there are twodistinct flow layers for the retentate and permeate/filtrate sides, butif the retentate port 604 resides on the retentate member ofdevice/module (that is, the cells are driven through the channel abovethe membrane and the filtrate (medium) passes to the portion of thechannel below the membrane), the permeate/filtrate port 606 will resideon the permeate member of device/module and vice versa (that is, if thecell sample is driven through the channel below the membrane, thefiltrate (medium) passes to the portion of the channel above themembrane). Due to the high pressures used to transfer the cell cultureand fluids through the flow channel of the TFF device, the effect ofgravity is negligible.

At the conclusion of a “pass” in either of the growth and concentrationprocesses, the cell sample is collected by passing through the retentateport 604 and into the retentate reservoir (not shown). To initiateanother “pass”, the cell sample is passed again through the TFF device,this time in a flow direction that is reversed from the first pass. Thecell sample is collected by passing through the retentate port 604 andinto retentate reservoir (not shown) on the opposite end of thedevice/module from the retentate port 604 that was used to collect cellsduring the first pass. Likewise, the medium/buffer that passes throughthe membrane on the second pass is collected through the permeate port606 on the opposite end of the device/module from the permeate port 606that was used to collect the filtrate during the first pass, or throughboth ports. This alternating process of passing the retentate (theconcentrated cell sample) through the device/module is repeated untilthe cells have been grown to a desired optical density, and/orconcentrated to a desired volume, and both permeate ports (i.e., ifthere are more than one) can be open during the passes to reduceoperating time. In addition, buffer exchange may be effected by adding adesired buffer (or fresh medium) to the cell sample in the retentatereservoir, before initiating another “pass”, and repeating this processuntil the old medium or buffer is diluted and filtered out and the cellsreside in fresh medium or buffer. Note that buffer exchange and cellgrowth may (and typically do) take place simultaneously, and bufferexchange and cell concentration may (and typically do) take placesimultaneously. For further information and alternative embodiments onTFFs see, e.g., U.S. Ser. No. 16/516,701, filed 5 Sep. 2019.

As an alternative to the TFF module described above, a cellconcentration module comprising a hollow filter may be employed.Examples of filters suitable for use in the present disclosure includemembrane filters, ceramic filters and metal filters. The filter may beused in any shape; the filter may, for example, be cylindrical oressentially flat. Preferably, the filter used is a membrane filter, mostpreferably a hollow fiber filter. The term “hollow fiber” is meant toinclude a tubular membrane. The internal diameter of the tube is atleast 0.1 mm, more preferably at least 0.5 mm, most preferably at least0.75 mm and preferably the internal diameter of the tube is at most 10mm, more preferably at most 6 mm, most preferably at most 1 mm. Filtermodules comprising hollow fibers are commercially available from variouscompanies, including G.E. Life Sciences (Marlborough, Mass.) andInnovaPrep (Drexel, Mo.). Specific examples of hollow fiber filtersystems that can be used, modified or adapted for use in the presentmethods and systems include, but are not limited to, U.S. Pat. Nos.9,738,918; 9,593,359; 9,574,977; 9,534,989; 9,446,354; 9,295,824;8,956,880; 8,758,623; 8,726,744; 8,677,839; 8,677,840; 8,584,536;8,584,535; and 8,110,112.

Nucleic Acid Assembly Module

Certain embodiments of the automated multi-module cell editinginstruments comprising FTEPs of the present disclosure optionallyinclude a nucleic acid assembly module. The nucleic acid assembly moduleis configured to accept and assemble the nucleic acids necessary to beporated into desired cells using the FTEP and to facilitate the desiredgenome editing events. In general, the term “vector” refers to a nucleicacid molecule capable of transporting a desired nucleic acid to which ithas been linked into a cell. Vectors include, but are not limited to,nucleic acid molecules that are single-stranded, double-stranded, orpartially double-stranded; nucleic acid molecules that include one ormore free ends, no free ends (e.g., circular); nucleic acid moleculesthat include DNA, RNA, or both; and other varieties of polynucleotidesknown in the art. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments canbe inserted, such as by standard molecular cloning techniques. Anothertype of vector is a viral vector, where virally-derived DNA or RNAsequences are present in the vector for packaging into a virus (e.g.retroviruses, replication defective retroviruses, adenoviruses,replication defective adenoviruses, and adeno-associated viruses). Viralvectors also include polynucleotides carried by a virus for transfectioninto a host cell. Certain vectors are capable of autonomous replicationin a host cell into which they are introduced (e.g. bacterial vectorshaving a bacterial origin of replication and episomal mammalianvectors). Other vectors (e.g., non-episomal mammalian vectors) areintegrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively-linked. Such vectors are referred toherein as “expression vectors” or “editing vectors.” Common expressionvectors of utility in recombinant DNA techniques are often in the formof plasmids. Additional vectors include fosmids, phagemids, BACs, YACs,and other synthetic chromosomes.

Recombinant expression vectors can include a nucleic acid in a formsuitable for transcription, and for some nucleic acid sequences,translation and expression of the nucleic acid in a host cell, whichmeans that the recombinant expression vectors include one or moreregulatory elements-which may be selected on the basis of the host cellsto be used for expression—that are operatively-linked to the nucleicacid sequence to be expressed. Within a recombinant expression vector,“operably linked” is intended to mean that the nucleotide sequence ofinterest is linked to the regulatory element(s) in a manner that allowsfor transcription, and for some nucleic acid sequences, translation andexpression of the nucleotide sequence (e.g., in an in vitrotranscription/translation system or in a host cell when the vector isintroduced into the host cell). Appropriate recombination and cloningmethods are disclosed in US Pub. No. 2004/0171156, the contents of whichare herein incorporated by reference in their entirety for all purposes.

In some embodiments, a regulatory element is operably linked to one ormore elements of a targetable nuclease system so as to drivetranscription, and for some nucleic acid sequences, translation andexpression of the one or more components of the targetable nucleasesystem.

In addition, the polynucleotide sequence encoding the nucleicacid-guided nuclease can be codon optimized for expression in particularcells, such as prokaryotic or eukaryotic cells. Eukaryotic cells can beyeast, fungi, algae, plant, animal, or human cells. Eukaryotic cells maybe those of or derived from a particular organism, such as a mammal,including but not limited to human, mouse, rat, rabbit, dog, ornon-human mammal including non-human primate. In addition oralternatively, a vector may include a regulatory element operably linkedto a polynucleotide sequence, which, when transcribed, forms a guideRNA.

The nucleic acid assembly module can be configured to perform a widevariety of different nucleic acid assembly techniques in an automatedfashion. Nucleic acid assembly techniques that can be performed in thenucleic acid assembly module of the disclosed automated multi-modulecell editing instruments include, but are not limited to, those assemblymethods that use restriction endonucleases, including PCR, BioBrickassembly (U.S. Pat. No. 9,361,427), Type IIS cloning (e.g., GoldenGateassembly, European Patent Application Publication EP 2 395 087 A1), andLigase Cycling Reaction (de Kok, ACS Synth Biol., 3(2):97-106 (2014);Engler, et al., PLoS One, 3(11):e3647 (2008); and U.S. Pat. No.6,143,527). In other embodiments, the nucleic acid assembly techniquesperformed by the disclosed automated multi-module cell editinginstruments are based on overlaps between adjacent parts of the nucleicacids, such as Gibson Assembly®, CPEC, SLIC, Ligase Cycling etc.Additional assembly methods include gap repair in yeast (Bessa, Yeast,29(10):419-23 (2012)), gateway cloning (Ohtsuka, Curr Pharm Biotechnol,10(2):244-51 (2009)); U.S. Pat. No. 5,888,732; and 6,277,608), andtopoisomerase-mediated cloning (Udo, PLoS One, 10(9):e0139349 (2015);and U.S. Pat. No. 6,916,632). These and other nucleic acid assemblytechniques are described, e.g., in Sands and Brent, Curr Protoc MolBiol., 113:3.26.1-3.26.20 (2016).

The nucleic acid assembly module is temperature controlled dependingupon the type of nucleic acid assembly used in the automatedmulti-module cell editing instrument. For example, when PCR is utilizedin the nucleic acid assembly module, the module includes a thermocyclingcapability allowing the temperatures to cycle between denaturation,annealing and extension steps. When single temperature assembly methods(e.g., isothermal assembly methods) are utilized in the nucleic acidassembly module, the module provides the ability to reach and hold atthe temperature that optimizes the specific assembly process beingperformed. These temperatures and the duration for maintaining thesetemperatures can be determined by a preprogrammed set of parametersexecuted by a script, or manually controlled by the user using theprocessing system of the automated multi-module cell editing instrument.

In one embodiment, the nucleic acid assembly module is a module toperform assembly using a single, isothermal reaction. Certain isothermalassembly methods can combine simultaneously up to 15 nucleic acidfragments based on sequence identity. The assembly method provides, insome embodiments, nucleic acids to be assembled which include anapproximate 20-40 base overlap with adjacent nucleic acid fragments. Thefragments are mixed with a cocktail of three enzymes—an exonuclease, apolymerase, and a ligase-along with buffer components. Because theprocess is isothermal and can be performed in a 1-step or 2-step methodusing a single reaction vessel, isothermal assembly reactions are idealfor use in an automated multi-module cell editing instrument. The 1-stepmethod allows for the assembly of up to five different fragments using asingle step isothermal process. The fragments and the master mix ofenzymes are combined and incubated at 50° C. for up to one hour. For thecreation of more complex constructs with up to fifteen fragments or forincorporating fragments from 100 bp up to 10 kb, typically the 2-step isused, where the 2-step reaction requires two separate additions ofmaster mix; one for the exonuclease and annealing step and a second forthe polymerase and ligation steps.

Cell Enrichment Module

One optional aspect of the present disclosure provides automated modulesand instruments for nucleic acid-guided nuclease genome editing thatimplement enrichment techniques for cells whose genomes have beenproperly edited. The enrichment module performs methods that use cellsingulation and normalization to reduce growth competition betweenedited and unedited cells or utilizes methods that take advantage ofinducing editing at a specific time during cell growth. Singulationovercomes growth bias from unedited cells or cells containing editsconferring growth advantages or disadvantages. The methods, modules andinstruments may be applied to all cell types including, archaeal,prokaryotic, and eukaryotic (e.g., yeast, fungal, plant and animal)cells.

Singulating or substantially singulating, induction of editing, andnormalization of cell colonies leads to 2-250×, 10-225×, 25-200×,40-175×, 50-150×. 60-100×, or 5-100× gains in identifying edited cellsover prior art methods and generates arrayed or pooled edited cellscomprising genome libraries. Additionally, the methods, modules, andinstruments may be leveraged to create iterative editing systems togenerate combinatorial libraries, identify rare cell edits, and enablehigh-throughput enrichment applications to identify editing activity.

The compositions and methods described herein improve nucleicacid-guided nuclease editing systems in which nucleic acid-guidednucleases (e.g., RNA-guided nucleases) are used to edit specific targetregions in an organism's genome. FIG. 7A depicts a solid wall device7050 and a workflow for singulating cells in microwells in the solidwall device, where in this workflow one or both of the gRNA and nucleaseare under the control of an inducible promoter. At the top left of thefigure (i), there is depicted solid wall device 7050 with microwells7052. A section 7054 of solid wall device 7050 is shown at (ii), alsodepicting microwells 7052. At (iii), a side cross-section of solid walldevice 7050 is shown, and microwells 7052 have been loaded, where, inthis embodiment, Poisson loading has taken place; that is, eachmicrowell has one (e.g., microwells 7052, 7056) or no cells, and thelikelihood that any one microwell has more than one cell is low. Note,however, that in alternative embodiments substantialsingulation-partitioning cells into small “groups” of less than 20 cellsper partition, and more preferably less than 10 cells per partition—maybe performed depending on the plexity of the library. At (iv), workflow7040 is illustrated where substrate 7050 having microwells 7052 showsmicrowells 7056 with one cell per microwell, microwells 7057 with nocells in the microwells, and one microwell 7060 with two cells in themicrowell. In step 7051, the cells in the microwells are allowed todouble approximately 2-50 times to form clonal colonies (v), thenediting is induced 7053 by heating the substrate (e.g., fortemperature-induced editing) or flowing chemicals under or over thesubstrate (e.g., sugars, antibiotics for chemical-induced editing) or bymoving the solid wall device to a different medium, which isparticularly facile if the solid wall device is placed on a fluidpermeable membrane which forms the bottom of microwells 7052. Afterinduction of editing 7053, many cells in the colonies of cells that havebeen edited die as a result of the double-strand cuts caused by activeediting, and there is possibly a lag in growth for the edited cells thatdo survive but must repair and recover following editing (microwells7058), where cells that do not undergo editing thrive (microwells 7059)(vi). All cells are allowed to grow to continue to establish coloniesand normalize, where the colonies of edited cells in microwells 7058catch up in size and/or cell number with the cells in microwells 7059that do not undergo editing (vii) due to cell senescence as the uneditedcells reach stationary phase. Once the cell colonies are normalized,either pooling of all cells in the microwells can take place, in whichcase the cells are enriched for edited cells by eliminating the biasfrom non-editing cells and fitness effects from editing; alternatively,colony growth in the microwells is monitored after editing, and slowgrowing colonies (e.g., the cells in microwells 7058) are identified andselected (e.g., “cherry picked”) resulting in even greater enrichment ofedited cells.

In growing the cells, the medium used will depend, of course, on thetype of cells being edited—e.g., bacterial, yeast or mammalian. Forexample, medium for bacterial growth includes LB, SOC, M9 Minimalmedium, and Magic medium; medium for yeast cell growth includes TPD,YPG, YPAD, and synthetic minimal medium; and medium for mammalian cellgrowth includes MEM, DMEM, IMDM, RPMI, and Hanks.

A module useful for performing the method depicted in FIG. 7A is a solidwall isolation, incubation, and normalization (SWIIN) module. FIG. 7Bdepicts an embodiment of a SWIIN module 750 from an exploded topperspective view. In SWIIN module 750 the retentate member is formed onthe bottom of a top of a SWIIN module component and the permeate memberis formed on the top of the bottom of a SWIIN module component.

The SWIIN module 750 in FIG. 7B comprises from the top down, a reservoirgasket or cover 758, a retentate member 704 (where a retentate flowchannel cannot be seen in this FIG. 7B), a perforated member 701 swagedwith a filter (filter not seen in FIG. 7B), a permeate member 708comprising integrated reservoirs (permeate reservoirs 752 and retentatereservoirs 754), and two reservoir seals 762, which seal the bottom ofpermeate reservoirs 752 and retentate reservoirs 754. A permeate channel760 a can be seen disposed on the top of permeate member 708, defined bya raised portion 776 of serpentine channel 760 a, and ultrasonic tabs764 can be seen disposed on the top of permeate member 708 as well. Theperforations that form the wells on perforated member 701 are not seenin this FIG. 7B; however, through-holes 766 to accommodate theultrasonic tabs 764 are seen. In addition, supports 770 are disposed ateither end of SWIIN module 750 to support SWIIN module 750 and toelevate permeate member 708 and retentate member 704 above reservoirs752 and 754 to minimize bubbles or air entering the fluid path from thepermeate reservoir to serpentine channel 760 a or the fluid path fromthe retentate reservoir to serpentine channel 760 b (neither fluid pathis seen in this FIG. 7B).

In this FIG. 7B, it can be seen that the serpentine channel 760 a thatis disposed on the top of permeate member 708 traverses permeate member708 for most of the length of permeate member 708 except for the portionof permeate member 708 that comprises permeate reservoirs 752 andretentate reservoirs 754 and for most of the width of permeate member708. As used herein with respect to the distribution channels in theretentate member or permeate member, “most of the length” means about95% of the length of the retentate member or permeate member, or about90%, 85%, 80%, 75%, or 70% of the length of the retentate member orpermeate member. As used herein with respect to the distributionchannels in the retentate member or permeate member, “most of the width”means about 95% of the width of the retentate member or permeate member,or about 90%, 85%, 80%, 75%, or 70% of the width of the retentate memberor permeate member.

In this embodiment of a SWIIN module, the perforated member includesthrough-holes to accommodate ultrasonic tabs disposed on the permeatemember. Thus, in this embodiment the perforated member is fabricatedfrom 316 stainless steel, and the perforations form the walls ofmicrowells while a filter or membrane is used to form the bottom of themicrowells. Typically, the perforations (microwells) are approximately150 μm-200 μm in diameter, and the perforated member is approximately125 μm deep, resulting in microwells having a volume of approximately2.5 nl, with a total of approximately 200,000 microwells. The distancebetween the microwells is approximately 279 μm center-to-center. Thoughhere the microwells have a volume of approximately 2.5 nl, the volume ofthe microwells may be from 1 to 25 nl, or preferably from 2 to 10 nl,and even more preferably from 2 to 4 nl. As for the filter or membrane,like the filter described previously, filters appropriate for use aresolvent resistant, contamination free during filtration, and are able toretain the types and sizes of cells of interest. For example, in orderto retain small cell types such as bacterial cells, pore sizes can be aslow as 0.10 μm, however for other cell types (e.g., such as formammalian cells), the pore sizes can be as high as 10.0 μm-20.0 μm ormore. Indeed, the pore sizes useful in the cell concentrationdevice/module include filters with sizes from 0.10 μm, 0.11 μm, 0.12 μm,0.13 μm, 0.14 μm, 0.15 μm, 0.16 μm, 0.17 μm, 0.18 μm, 0.19 μm, 0.20 μm,0.21 μm, 0.22 μm, 0.23 μm, 0.24 μm, 0.25 μm, 0.26 μm, 0.27 μm, 0.28 μm,0.29 μm, 0.30 μm, 0.31 μm, 0.32 μm, 0.33 μm, 0.34 μm, 0.35 μm, 0.36 μm,0.37 μm, 0.38 μm, 0.39 μm, 0.40 μm, 0.41 μm, 0.42 μm, 0.43 μm, 0.44 μm,0.45 μm, 0.46 μm, 0.47 μm, 0.48 μm, 0.49 μm, 0.50 μm and larger. Thefilters may be fabricated from any suitable material including cellulosemixed ester (cellulose nitrate and acetate) (CME), polycarbonate (PC),polyvinylidene fluoride (PVDF), polyethersulfone (PES),polytetrafluoroethylene (PTFE), nylon, or glass fiber.

The cross-section configuration of the mated serpentine channel may beround, elliptical, oval, square, rectangular, trapezoidal, or irregular.If square, rectangular, or another shape with generally straight sides,the cross section may be from about 2 mm to 15 mm wide, or from 3 mm to12 mm wide, or from 5 mm to 10 mm wide. If the cross section of themated serpentine channel is generally round, oval or elliptical, theradius of the channel may be from about 3 mm to 20 mm in hydraulicradius, or from 5 mm to 15 mm in hydraulic radius, or from 8 mm to 12 mmin hydraulic radius.

Serpentine channels 760 a and 760 b can have approximately the samevolume or the serpentine channels 760 a and 760 b may have differentvolumes. For example, each “side” or portion 760 a, 760 b of theserpentine channel may have a volume of, e.g., 2 mL, or serpentinechannel 760 a of permeate member 708 may have a volume of 2 mL, and theserpentine channel 760 b of retentate member 704 may have a volume of,e.g., 3 mL. The volume of fluid in the serpentine channel may range fromabout 2 mL to about 80 mL, or about 4 mL to 60 mL, or from 5 mL to 40mL, or from 6 mL to 20 mL (note these volumes apply to a SWIIN modulecomprising a, e.g., 50-500K perforation member). The volume of thereservoirs may range from 5 mL to 50 mL, or from 7 mL to 40 mL, or from8 mL to 30 mL or from 10 mL to 20 mL, and the volumes of all reservoirsmay be the same or the volumes of the reservoirs may differ (e.g., thevolume of the permeate reservoirs is greater than that of the retentatereservoirs).

The serpentine channel portions 760 a and 760 b of the permeate member708 and retentate member 704, respectively, are approximately 200 mmlong, 130 mm wide, and 4 mm thick, though in other embodiments, theretentate and permeate members can be from 75 mm to 400 mm in length, orfrom 100 mm to 300 mm in length, or from 150 mm to 250 mm in length;from 50 mm to 250 mm in width, or from 75 mm to 200 mm in width, or from100 mm to 150 mm in width; and from 2 mm to 15 mm in thickness, or from4 mm to 10 mm in thickness, or from 5 mm to 8 mm in thickness.Embodiments the retentate (and permeate) members may be fabricated fromPMMA (poly(methyl methacrylate) or other materials may be used,including polycarbonate, cyclic olefin co-polymer (COC), glass,polyvinyl chloride, polyethylene, polyamide, polypropylene, polysulfone,polyurethane, and co-polymers of these and other polymers. Preferably atleast the retentate member is fabricated from a transparent material sothat the cells can be visualized (see, e.g., FIG. 7E and the descriptionthereof). For example, a video camera may be used to monitor cell growthby, e.g., density change measurements based on an image of an emptywell, with phase contrast, or if, e.g., a chromogenic marker, such as achromogenic protein, is used to add a distinguishable color to thecells. Chromogenic markers such as blitzen blue, dreidel teal, virginiaviolet, vixen purple, prancer purple, tinsel purple, maccabee purple,donner magenta, cupid pink, seraphina pink, scrooge orange, and leororange (the Chromogenic Protein Paintbox, all available from ATUM(Newark, Calif.)) obviate the need to use fluorescence, althoughfluorescent cell markers, fluorescent proteins, and chemiluminescentcell markers may also be used.

Because the retentate member preferably is transparent, colony growth inthe SWIIN module can be monitored by automated devices such as thosesold by JoVE (ScanLag™ system, Cambridge, Mass.) (also seeLevin-Reisman, et al., Nature Methods, 7:737-39 (2010)). Cell growthfor, e.g., mammalian cells may be monitored by, e.g., the growth monitorsold by IncuCyte (Ann Arbor, Mich.) (see also, Choudhry, PLos One,11(2):e0148469 (2016)). Further, automated colony pickers may beemployed, such as those sold by, e.g., TECAN (Pickolo™ system,Mannedorf, Switzerland); Hudson Inc. (RapidPick™, Springfield, N.J.);Molecular Devices (QPix 400 TMsystem, San Jose, Calif.); and SingerInstruments (PIXL™ system, Somerset, UK).

Due to the heating and cooling of the SWIIN module, condensation mayaccumulate on the retentate member which may interfere with accuratevisualization of the growing cell colonies. Condensation of the SWIINmodule 750 may be controlled by, e.g., moving heated air over the top of(e.g., retentate member) of the SWIIN module 750, or by applying atransparent heated lid over at least the serpentine channel portion 760b of the retentate member 704. See, e.g., FIG. 7E and the descriptionthereof infra.

In SWIIN module 750 cells and medium at a dilution appropriate forPoisson or substantial Poisson distribution of the cells in themicrowells of the perforated member—are flowed into serpentine channel760 b from ports in retentate member 704, and the cells settle in themicrowells while the medium passes through the filter into serpentinechannel 760 a in permeate member 708. The cells are retained in themicrowells of perforated member 701 as the cells cannot travel throughfilter 703. Appropriate medium may be introduced into permeate member708 through permeate ports 711. The medium flows upward through filter703 to nourish the cells in the microwells (perforations) of perforatedmember 701. Additionally, buffer exchange can be effected by cyclingmedium through the retentate and permeate members. In operation, thecells are deposited into the microwells, are grown for an initial, e.g.,2-100 doublings, editing is induced by, e.g., raising the temperature ofthe SWIIN to 42° C. to induce a temperature inducible promoter or byremoving growth medium from the permeate member and replacing the growthmedium with a medium comprising a chemical component that induces aninducible promoter.

Once editing has taken place, the temperature of the SWIIN may bedecreased, or the inducing medium may be removed and replaced with freshmedium lacking the chemical component thereby de-activating theinducible promoter. The cells then continue to grow in the SWIIN module750 until the growth of the cell colonies in the microwells isnormalized. For the normalization protocol, once the colonies arenormalized, the colonies are flushed from the microwells by applyingfluid or air pressure (or both) to the permeate member serpentinechannel 760 a and thus to filter 703 and pooled. Alternatively, ifcherry picking is desired, the growth of the cell colonies in themicrowells is monitored, and slow-growing colonies are directlyselected; or, fast-growing colonies are eliminated.

FIG. 7C is a top perspective view of a SWIIN module with the retentateand perforated members in partial cross section. In this FIG. 7C, it canbe seen that serpentine channel 760 a is disposed on the top of permeatemember 708 is defined by raised portions 776 and traverses permeatemember 708 for most of the length and width of permeate member 708except for the portion of permeate member 708 that comprises thepermeate and retentate reservoirs (note only one retentate reservoir 752can be seen). Moving from left to right, reservoir gasket 758 isdisposed upon the integrated reservoir cover 778 (cover not seen in thisFIG. 7C) of retentate member 704. Gasket 758 comprises reservoir accessapertures 732 a, 732 b, 732 c, and 732 d, as well as pneumatic ports 733a, 733 b, 733 c and 733 d. Also at the far left end is support 770.Disposed under permeate reservoir 752 can be seen one of two reservoirseals 762. In addition to the retentate member being in cross section,the perforated member 701 and filter 703 (filter 703 is not seen in thisFIG. 7C) are in cross section. Note that there are a number ofultrasonic tabs 764 disposed at the right end of SWIIN module 750 and onraised portion 776 which defines the channel turns of serpentine channel760 a, including ultrasonic tabs 764 extending through through-holes 766of perforated member 701. There is also a support 770 at the end distalreservoirs 752, 754 of permeate member 708.

FIG. 7D is a side perspective view of an assembled SWIIIN module 750,including, from right to left, reservoir gasket 758 disposed uponintegrated reservoir cover 778 (not seen) of retentate member 704.Gasket 758 may be fabricated from rubber, silicone, nitrile rubber,polytetrafluoroethylene, a plastic polymer such aspolychlorotrifluoroethylene, or other flexible, compressible material.Gasket 758 comprises reservoir access apertures 732 a, 732 b, 732 c, and732 d, as well as pneumatic ports 733 a, 733 b, 733 c and 733 d. Also atthe far-left end is support 770 of permeate member 708. In addition,permeate reservoir 752 can be seen, as well as one reservoir seal 762.At the far-right end is a second support 770.

Imaging of cell colonies growing in the wells of the SWIIN is desired inmost implementations for, e.g., monitoring both cell growth and deviceperformance and imaging is necessary for cherry-picking implementations.Real-time monitoring of cell growth in the SWIIN requires backlighting,retentate plate (top plate) condensation management and a system-levelapproach to temperature control, air flow, and thermal management. Insome implementations, imaging employs a camera or CCD device withsufficient resolution to be able to image individual wells. For example,in some configurations a camera with a 9-pixel pitch is used (that is,there are 9 pixels center-to-center for each well). Processing theimages may, in some implementations, utilize reading the images ingrayscale, rating each pixel from low to high, where wells with no cellswill be brightest (due to full or nearly-full light transmission fromthe backlight) and wells with cells will be dim (due to cells blockinglight transmission from the backlight). After processing the images,thresholding is performed to determine which pixels will be called“bright” or “dim”, spot finding is performed to find bright pixels andarrange them into blocks, and then the spots are arranged on a hexagonalgrid of pixels that correspond to the spots. Once arranged, the measureof intensity of each well is extracted, by, e.g., looking at one or morepixels in the middle of the spot, looking at several to many pixels atrandom or pre-set positions, or averaging X number of pixels in thespot. In addition, background intensity may be subtracted. Thresholdingis again used to call each well positive (e.g., containing cells) ornegative (e.g., no cells in the well). The imaging information may beused in several ways, including taking images at time points formonitoring cell growth. Monitoring cell growth can be used to, e.g.,remove the “muffin tops” of fast-growing cells followed by removal ofall cells or removal of cells in “rounds” as described above, or recovercells from specific wells (e.g., slow-growing cell colonies);alternatively, wells containing fast-growing cells can be identified andareas of UV light covering the fast-growing cell colonies can beprojected (or rastered with shutters) onto the SWIIN to irradiate orinhibit growth of those cells. Imaging may also be used to assure properfluid flow in the serpentine channel 760.

FIG. 7E depicts the embodiment of the SWIIN module in FIGS. 7B-7Dfurther comprising a heat management system including a heater and aheated cover. The heater cover facilitates the condensation managementthat is required for imaging. Assembly 798 comprises a SWIIN module 750seen lengthwise in cross section, where one permeate reservoir 752 isseen. Disposed immediately upon SWIIN module 750 is cover 794 anddisposed immediately below SWIIN module 750 is backlight 780, whichallows for imaging. Beneath and adjacent to the backlight and SWIINmodule is insulation 782, which is disposed over a heatsink 784. In thisFIG. 7E, the fins of the heatsink would be in-out of the page. Inaddition there is also axial fan 786 and heat sink 788, as well as twothermoelectric coolers 792, and a controller 790 to control thepneumatics, thermoelectric coolers, fan, solenoid valves, etc. Thearrows denote cool air coming into the unit and hot air being removedfrom the unit. It should be noted that control of heating allows forgrowth of many different types of cells (prokaryotic and eukaryotic) aswell as strains of cells that are, e.g., temperature sensitive, etc.,and allows use of temperature-sensitive promoters. Temperature controlallows for protocols to be adjusted to account for differences intransformation efficiency, cell growth and viability. For more detailsregarding solid wall isolation incubation and normalization devices seeU.S. Ser. No. 16/399,988, filed 30 Apr. 2019; Ser. No. 16/454,865, filed26 Jun. 2019; Ser. No. 16/540,606, filed 14 Aug. 2019; Ser. No.16/597,826, filed 9 Oct. 2019; and Ser. No. 16/597,831, filed 9 Oct.2019. For alternative isolation, incubation and normalization modules,see U.S. Ser. No. 16/536,049, filed 8 Aug. 2019.

Use of the Cell Growth Device

FIG. 8 is a flow chart of an example method 800 for using an automatedmulti-module cell editing instrument such as the systems illustrated inFIGS. 4A-4C which include the FTEP devices described in relation toFIGS. 1A-1P. A processing system, for example, directs the processingstage of the method 800. For example, a software script may identifysettings for each processing stage and instructions for movement of arobotic handling system to perform the actions of the method 800. Insome embodiments, a software instruction script may be identified by areagent cartridge supplied to the automated multi-module cell editinginstrument. For example, the reagent cartridge may includemachine-readable indicia, such as a bar code or QR code, includingidentification of a script stored in a memory of the automatedmulti-module cell editing instrument. In another example, the reagentcartridge may contain a downloadable script embedded in machine-readableindicia such as a radio frequency (RF) tag. In other embodiments, theuser may identify a script, for example through downloading the scriptvia a wired or wireless connection to the processing system of theautomated multi-module cell editing instrument or through selecting astored script through a user interface of the automated multi-modulecell editing instrument. In a particular example, the automatedmulti-module cell editing instrument may include a touch screeninterface for submitting user settings and activating cell processing.Again, the automated multi-module cell processing instrument is astand-alone instrument, and between the script, reagent reservoirs, andliquid handling system facilitates live cell editing in an entirelyautomated manner without human intervention.

In some implementations, the method 800 begins with transferring cellsto a cell growth module (802). The growth module may be any growthmodule amendable to automation such as, for example, the cell growthmodule 550 described in relation to FIGS. 5B-5D. In a particularexample, the processing system may direct the robotic handling system totransfer cells to the growth module. In another example, the cells maybe transferred from a reagent cartridge to the growth module by therobotic handling system. In some embodiments, the growth vial maycontain growth media and be supplied, e.g., as part of a kit. In otherembodiments, the growth vial may be filled with medium transferred,e.g., via the liquid handling device, from a reagent container.

In some embodiments, prior to transferring the cells (e.g., from thereagent cartridge or from a vial added to the instrument),machine-readable indicia may be scanned upon the vial or other containersituated in a position designated for cells to confirm that the vial orcontainer is marked as containing cells. Further, the machine-readableindicia may indicate a type of cells provided to the instrument. Thetype of cells, in some embodiments, may cause the instrument to select aparticular processing script (e.g., series of instructions for therobotic handling system and settings and activation of the variousmodules).

In some implementations, the cells are grown in the growth module to adesired optical density (804). For example, the processing system maymanage a temperature setting of the growth module for incubating thecells during the growth cycle. The processing system may further receivesensor signals from the growth module indicative of optical density andanalyze the sensor signals to monitor growth of the cells. In someembodiments, a user may set growth parameters for managing growth of thecells. For example, temperature, and the degree of agitation of thecells. Further, in some embodiments, the user may be updated regardingthe growth process. The updates, in some examples, may include a messagepresented on a user interface of the automated multi-module cell editinginstrument, a text message to a user's cell phone number, an emailmessage to an email account, or a message transmitted to an appexecuting upon a portable electronic device (e.g., cell phone, tablet,etc.). Responsive to the messages, in some embodiments, the user maymodify parameters, such as temperature, to adjust cell growth. Forexample, the user may submit updated parameters through a user interfaceof the automated multi-module cell editing instrument or through aportable computing device application in communication with theautomated multi-module cell editing instrument, such as a user interface(see, e.g., touch screen display 401 of FIG. 4C).

Although described in relation to optical density, in otherimplementations cell growth within the growth module may be monitoredusing a different measure of cell density and physiological state suchas, in some examples, pH, dissolved oxygen, released enzymes, acousticproperties, and electrical properties.

In some implementations, upon reaching the desired optical density(804), the cells are transferred from the growth module to a filtrationmodule or cell wash and concentration module (806). The robotic handlingsystem, for example, may transfer the cells from the growth module tothe cell concentration module. The cell concentration module, forexample, may be (and typically is) designed to render the cellselectrocompetent. See FIGS. 6A-6D in relation to the TFF device, above.The cells are rendered electrocompetent and eluted in the filtrationmodule or cell wash and concentration module (808). The cells may beeluted using a wash solution. For example, the cells may be eluted usingreagents from a reagent supply.

Once the cells have been rendered electrocompetent and suspended in anappropriate volume such as 50 μL to 10 mL, or 100 μL to 9 mL, or 150 μLto 8 mL, or 250 μL to 7 mL, or 500 μL to 6 mL, or 750 μL to 5 mL fortransformation (808), the cells are transferred to, e.g., an FTEP module(812). The robotic handling system, for example, may transfer the cellsfrom the cell concentration device or module to the FTEP 812. Thefiltration module may be physically coupled to the FTEP device, or thesemodules may be separate.

In some implementations, nucleic acids are prepared outside of theautomated multi-module cell editing instrument. For example, anassembled vector or other nucleic acid assembly may be included as areagent in, e.g., a reagent cartridge 810 by a user prior to running thetransformation process and other processes in the method 800. Ifprovided in a reagent cartridge, the nucleic acid assembly (e.g.,editing vector library) is transferred to the FTEP device as well.

The cells are transformed in the FTEP module with the editing vectorprovided in the reagent cartridge (814). A buffer or medium may betransferred to the transformation module and added to the cells so thatthe cells may be suspended in a buffer or medium that is favorable forcell survival during electroporation. Prior to transferring the bufferor medium, machine-readable indicia may be scanned upon the vial orother container or reservoir situated in the position designated for thebuffer or medium to confirm the contents of the vial, container, orreservoir. Further, the machine-readable indicia may indicate a type ofbuffer or medium provided to the instrument. The type of buffer ormedium, in some embodiments, may cause the instrument to select aparticular processing script (e.g., settings and activation of thetransformation module appropriate for the particular buffer or medium).For bacterial cell electroporation, low conductance mediums, such aswater or glycerol solutions, may be used to reduce the heat productionby transient high current. For yeast cells, a sorbitol solution may beused. For mammalian cell electroporation, cells may be suspended in ahighly conductive medium or buffer, such as MEM, DMEM, IMDM, RPMI,Hanks', PBS, HBSS, HeBS and Ringer's solution. In a particular example,the robotic handling system may transfer a buffer solution to FTEPmodule from the reagent cartridge. As described in relation to FIGS.1A-1P and 3A-3F, the FTEP device may be a disposable FTEP device and/orthe FTEP device may be provided as part of the reagent cartridge.Alternatively, the FTEP device may a separate module.

Once transformed, the cells are transferred to, e.g., a secondgrowth/recovery/editing module (816) such as the cell growth moduledescribed in relation to FIGS. 5A-5D. The robotic handling system, forexample, may transfer the transformed cells to the second growth modulethrough a sipper or pipettor interface. In another example, the robotichandling system may transfer a vial containing the transformed cellsfrom a chamber of the transformation module to a chamber of the secondgrowth module.

The second growth module, in some embodiments, acts as a recoverymodule, allowing the cells to recover from the transformation process.In other embodiments, the cells may be provided to a separate recoverymodule prior to being transported to the second growth module. Duringrecovery, the second growth module allows the transformed cells touptake and, in certain aspects, integrate the introduced nucleic acidsinto the genome of the cell. The second growth module may be configuredto incubate the cells at any user-defined temperature optimal for cellgrowth, preferably 25°, 30°, or 37° C.

In some embodiments, the second growth module behaves as a selectionmodule, selecting the transformed cells based on an antibiotic or otherreagent. In one example, the RNA-guided nuclease (RGN) protein system isused for selection to cleave the genomes of cells that have not receivedthe desired edit. In the example of an antibiotic selection agent, theantibiotic may be added to the second growth module to enact selection.Suitable antibiotic resistance genes include, but are not limited to,genes such as ampicillin-resistance gene, tetracycline-resistance gene,kanamycin-resistance gene, neomycin-resistance gene,canavanine-resistance gene, blasticidin-resistance gene,hygromycin-resistance gene, puromycin-resistance gene, orchloramphenicol-resistance gene. The robotic handling system, forexample, may transfer the antibiotic to the second growth module througha sipper or pipettor interface. In some embodiments, removing dead cellbackground is aided by using lytic enhancers such as detergents, osmoticstress by hyponic wash, temperature, enzymes, proteases, bacteriophage,reducing agents, or chaotropes. The processing system, for example, mayalter environmental variables, such as temperature, to induce selection,while the robotic handling system may deliver additional materials(e.g., detergents, enzymes, reducing agents, etc.) to aid in selection.In other embodiments, cell removal and/or media exchange by filtrationis used to reduce dead cell background.

In further embodiments, in addition to or as an alternative to applyingselection, the second growth module serves as an editing module,allowing for genome editing in the transformed cells. Alternatively, inother embodiments, the cells post-recovery and post-selection (ifperformed) are transferred to a separate editing module. As an editingmodule, the second growth module induces editing of the cells' genomes,e.g., through facilitating expression of the introduced nucleic acids.Expression of the nuclease and/or editing cassette nucleic acids mayinvolve one or more of chemical, light, viral, or temperature inductionmethods. The second growth module, for example, may be configured toheat or cool the cells during a temperature induction process. In aparticular illustration, the cells may be induced by heating at 42°C.−50° C. Further to the illustration, the cells may then be cooled to0-10° C. after induction. In the example of chemical or viral induction,an inducing agent may be transferred to the second growth module toinduce editing. If an inducible nuclease and/or editing cassette wasintroduced to the cells during editing, it can be induced throughintroduction of an inducer molecule. The inducing agent or inducermolecule, in some implementations, is transferred to the second growthmodule by the robotic handling system, e.g., through a pipettor orsipper interface.

In some implementations, if no additional cell editing is desired (818),the cells may be transferred from the cell growth module to a storageunit for later removal from the automated multi-module cell editinginstrument (820). The robotic handling system, for example, may transferthe cells to a storage unit through a sipper or pipettor interface. Inanother example, the robotic handling system may transfer a vialcontaining the cells from a chamber of the second growth module to avial or tube within the storage unit.

In some implementations, if additional cell editing is desired (818),the cells may be transferred to a growth module (802), grown to adesired OD (804), transferred to a cell concentration module (806), thenconcentrated and rendered electrocompetent (808). Further, in someembodiments, a new assembled nucleic acid sample may be prepared by thenucleic acid assembly module at this time, or, alternatively, a secondfully assembled nucleic acid may be directly introduced to the cellsfrom, e.g., the reagent cartridge. Prior to recursive editing, in someembodiments, the automated multi-module cell editing instrument mayrequire additional materials be supplied by the user, e.g., through theintroduction of one or more separate reagents vials or cartridge.

The steps may be the same or different during the second round ofediting. For example, in some embodiments, upon a subsequent executionof step 804, a selective growth medium is transferred to the growthmodule to enable selection of edited cells from the first round ofediting. The robotic handling system may transfer the selective growthmedium from a vial or container in a reagent cartridge situated in aposition designated for selective growth medium. Prior to transferringthe selective growth medium, machine-readable indicia may be scannedupon the vial or other container or reservoir situated in the positiondesignated for the selective growth medium to confirm the contents ofthe vial, container, or reservoir. Further, the machine-readable indiciamay indicate a type of selective growth medium provided to theinstrument. The type of selective growth medium, in some embodiments,may cause the instrument to select a particular processing script (e.g.,settings and activation of the growth module appropriate for theparticular selective growth medium). Particular examples of recursiveediting workflows are described in relation to FIG. 10.

In some implementations, the method 800 can be timed to introducematerials and/or complete the editing cycle or growth cycle incoordination with a user's schedule. For example, the automatedmulti-module cell editing instrument may provide the user the ability toschedule completion of one or more cell processing cycles (e.g., one ormore recursive edits) such that the method 800 is enacted with a goal ofcompletion at the user's preferred time. The time scheduling, forexample, may be set through a user interface. For illustration only, auser may set completion of a first cycle to 4:00 PM so that the user cansupply additional cartridges of materials to the automated multi-modulecell editing instrument to enable overnight processing of another roundof cell editing. Thus, a user may time the programs so that two or morecycles may be programmed in a specific time period, e.g., a 24-hourperiod.

In some implementations, throughout the method 800, the automatedmulti-module cell editing instrument may alert the user to its currentstatus. For example, the user interface may present a graphicalindication of the present stage of processing. In a particular example,a front face of the automated multi-module call processing instrumentmay be overlaid with a user interface (e.g., touch screen) that presentsan animated graphic depicting present status of the cell processing. Theuser interface may further present any user and/or default settingsassociated with the current processing stage (e.g., temperature setting,time setting, etc.). In certain implementations, the status may becommunicated to a user via a wireless communications controller.

Although illustrated as a particular series of operations, in otherembodiments, more or fewer steps may be included in the method 800. Forexample, in some embodiments, prior to engaging in each round ofediting, the contents of reservoirs, reagent cartridges, and/or vialsmay be screened to confirm appropriate materials are available toproceed with processing. For example, in some embodiments, one or moreimaging sensors (e.g., barcode scanners, cameras, etc.) may confirmcontents at various locations within the housing of the automatedmulti-module cell editing instrument. In one example, multiple imagingsensors may be disposed within the housing of the automated multi-modulecell editing instrument, each imaging sensor configured to detect one ormore materials (e.g., machine-readable indicia such as barcodes or QRcodes, shapes/sizes of materials, etc.). In another example, at leastone imaging sensor may be moved by the robotic handling system tomultiple locations to detect one or more materials. In furtherembodiments, one or more weight sensors may detect presence or absenceof disposable or replaceable materials. In an illustrative example, thetransfer tip supply holder may include a weight sensor to detect whetheror not tips have been loaded into the region. In another illustrativeexample, an optical sensor may detect that a level of liquid waste hasreached a threshold level, requiring disposal prior to continuation ofcell processing or addition of liquid if the minimum level has not beenreached to proceed. Requests for additional materials, removal of wastesupplies, or other user interventions (e.g., manual cleaning of one ormore elements, etc.), in some implementations, are presented on agraphical user interface of the automated multi-module cell editinginstrument. The automated multi-module cell editing instrument, in someimplementations, contacts the user with requests for new materials orother manual interventions, for example, through a software app, email,or text message.

FIG. 9 is a simplified block diagram of an embodiment of an exemplaryautomated multi-module cell processing instrument 900 comprising asingulation/growth/editing/normalization module 940 for enrichment foredited cells. The cell processing instrument 900 may include a housing944, a reservoir of cells to be transformed or transfected 902, and agrowth module (a cell growth device) 904. The cells to be transformedare transferred from a reservoir 902 to the growth module 904 to becultured until the cells hit a target OD. Once the cells hit the targetOD, the growth module 904 may cool or freeze the cells for laterprocessing, or the cells may be transferred to a cell concentrationmodule 930 where the cells are rendered electrocompetent andconcentrated to a volume optimal for cell transformation. Onceconcentrated, the cells are then transferred to the flow-throughelectroporation module 905 (e.g., transformation/transfection module).

In addition to the reservoir 902 for storing the cells, the automatedmulti-module cell processing instrument 900 may include a reservoir forstoring editing oligonucleotide cassettes 916 and a reservoir forstoring an expression vector backbone 918. Both the editingoligonucleotide cassettes and the expression vector backbone aretransferred from the reagent cartridge to a nucleic acid assembly module920, where the editing oligonucleotide cassettes are inserted into theexpression vector backbone. The assembled nucleic acids may betransferred into an optional purification module 922 for desaltingand/or other purification and/or concentration procedures needed toprepare the assembled nucleic acids for transformation. Alternatively,pre-assembled nucleic acids, e.g., an editing vector, may be storedwithin reservoir 916 or 918. Once the processes carried out by thepurification module 922 are complete, the assembled nucleic acids aretransferred to, e.g., an electroporation device 905, which alreadycontains the cell culture grown to a target OD and renderedelectrocompetent via cell concentration module 1130. In electroporationdevice 905, the assembled nucleic acids are introduced into the cells.Following electroporation, the cells are transferred into a combinedrecovery/dilution/selection module 910.

Following recovery, and, optionally, selection, the cells aretransferred to a singulation, selection, growth, induction, editing, andnormalization module 940, where the cells are diluted andcompartmentalized such that there is an average of one cell percompartment. Once singulated, the cells grown in, e.g., selectivemedium, for a pre-determined number of doublings. Once these initialcolonies are established, editing is induced and the edited cells areallowed to establish colonies, which are grown to terminal size (e.g.,the colonies are normalized). In some embodiments, editing is induced byone or more of the editing components being under the control of aninducible promoter. In some embodiments, the inducible promoter isactivated by a rise in temperature and “deactivated” by lowering thetemperature. Alternatively, in embodiments where the singulation deviceis a solid wall device comprising a filter forming the bottom of themicrowell, the solid wall device can be transferred to a plate (e.g.,agar plate or even to liquid medium) comprising a medium with acomponent that activates induced editing, then transferred to a mediumthat deactivates editing. Once the colonies are grown to terminal size,the colonies are pooled. Again, singulation overcomes growth bias fromunedited cells and growth bias resulting from fitness effects ofdifferent edits.

The recovery, dilution, selection, singulation, induction, editing andgrowth modules may all be separate, may be arranged and combined asshown in FIG. 9, or may be arranged or combined in other configurations.In certain embodiments, all of recovery, selection, singulation, growth,editing, and normalization are performed in a solid wall device.Alternatively, recovery, selection, and dilution, if necessary, areperformed in liquid medium in a separate vessel (module), thentransferred to the solid wallsingulation/growth/induction/editing/normalization module.

Once the normalized cell colonies are pooled, the cells may be stored,e.g., in a storage module 912, where the cells can be kept at, e.g., 4°C. until the cells are retrieved 914 for further study. Alternatively,the cells may be used in another round of editing. The multi-module cellprocessing instrument 900 is controlled by a processor 942 configured tooperate the instrument 900 based on user input, as directed by one ormore scripts, or as a combination of user input or a script. Theprocessor 942 may control the timing, duration, temperature, andoperations of the various modules of the instrument 900 and thedispensing of reagents. For example, the processor 942 may cool thecells post-transformation until editing is desired, upon which time thetemperature may be raised to a temperature conducive of genome editingand cell growth. The processor may be programmed with standard protocolparameters from which a user may select, a user may specify one or moreparameters manually, or one or more scripts associated with the reagentcartridge may specify one or more operations and/or reaction parameters.In addition, the processor 942 may notify the user (e.g., via anapplication to a smart phone or other device) that the cells havereached the target OD as well as update the user as to the progress ofthe cells in the various modules in the multi-module cell processinginstrument 900.

The automated multi-module cell processing instrument 900 is anuclease-directed genome editing system and can be used in singleediting systems (e.g., introducing one or more edits to a cellulargenome in a single editing process). The system of FIG. 10, describedbelow, is configured to perform sequential editing, e.g., usingdifferent nuclease-directed systems sequentially to provide two or moregenome edits in a cell; and/or recursive editing, e.g. utilizing asingle nuclease-directed system to introduce sequentially two or moregenome edits in a cell.

FIG. 10 illustrates another embodiment of a multi-module cell processinginstrument 1000. This embodiment depicts an exemplary system thatperforms recursive gene editing on a cell population. As with theembodiment shown in FIG. 9, the cell processing instrument 1000 mayinclude a housing 1044, a reservoir for storing cells to be transformedor transfected 1002, and a cell growth module (comprising, e.g., arotating growth vial) 1004. The cells to be transformed are transferredfrom a reservoir to the cell growth module 1004 to be cultured until thecells hit a target OD. Once the cells hit the target OD, the growthmodule may cool or freeze the cells for later processing or transfer thecells to a cell concentration module 1060 where the cells are subjectedto buffer exchange and rendered electrocompetent, and the volume of thecells may be reduced substantially. Once the cells have beenconcentrated to an appropriate volume, the cells are transferred toelectroporation device or module 1008. In addition to the reservoir forstoring cells, the multi-module cell processing instrument 1000 includesa reservoir for storing the vector pre-assembled with editingoligonucleotide cassettes 1052. The pre-assembled nucleic acid vectorsare transferred to the electroporation device 1008, which alreadycontains the cell culture grown to a target OD. In the electroporationdevice 1008, the nucleic acids are electroporated into the cells.Following electroporation, the cells are transferred into an optionalrecovery (and optionally, dilution) module 1056, where the cells areallowed to recover briefly post-transformation.

After recovery, the cells may be transferred to a storage module 1012,where the cells can be stored at, e.g., 4° C. for later processing, orthe cells may be diluted and transferred to aselection/growth/induction/editing module/device 1058. The cells areallowed to grow and editing is then induced by providing conditions(e.g., temperature, addition of an inducing or repressing chemical) toinduce editing. Note that the selection/growth/induction and editingmodules may be the same module or device, where all processes areperformed in, e.g., a solid wall singulation device, or selection and/ordilution may take place in a separate vessel before the cells aretransferred to an induction/editing module. As an alternative tosingulation in, e.g., a solid wall device, the transformed cells may begrown in—and editing can be induced in-bulk liquid (see, e.g., U.S. Ser.No. 16/545,097, filed 20 Aug. 2019. Once the putatively-edited cells arepooled, they may be subjected to another round of editing, beginningwith growth, cell concentration and treatment to renderelectrocompetent, and transformation by yet another donor nucleic acidin another editing cassette via the electroporation device/module 1008.

In electroporation device 1008, the cells selected from the first roundof editing are transformed by a second set of editing oligos (or othertype of oligos) and the cycle is repeated until the cells have beentransformed and edited by a desired number of, e.g., editing cassettes.The multi-module cell processing instrument 1000 exemplified in FIG. 10is controlled by a processor 1042 configured to operate the instrumentbased on user input or is controlled by one or more scripts including atleast one script associated with the reagent cartridge. The processor1042 may control the timing, duration, and temperature of variousprocesses, the dispensing of reagents, and other operations of thevarious modules of the instrument 1000. For example, a script or theprocessor may control the dispensing of cells, reagents, vectors, andediting oligonucleotides; which editing oligonucleotides are used forcell editing and in what order; the time, temperature and otherconditions used in the recovery and expression module, the wavelength atwhich OD is read in the cell growth module, the target OD to which thecells are grown, and the target time at which the cells will reach thetarget OD. In addition, the processor may be programmed to notify a user(e.g., via an application) as to the progress of the cells in theautomated multi-module cell processing instrument.

It should be apparent to one of ordinary skill in the art given thepresent disclosure that the process described may be recursive andmultiplexed; that is, cells may go through the workflow described inrelation to FIG. 10, then the resulting edited culture may go throughanother (or several or many) rounds of additional editing (e.g.,recursive editing) with different editing vectors. For example, thecells from round 1 of editing may be diluted and an aliquot of theedited cells edited by editing vector A may be combined with editingvector B, an aliquot of the edited cells edited by editing vector A maybe combined with editing vector C, an aliquot of the edited cells editedby editing vector A may be combined with editing vector D, and so on fora second round of editing. After round two, an aliquot of each of thedouble-edited cells may be subjected to a third round of editing, where,e.g., aliquots of each of the AB-, AC-, AD-edited cells are combinedwith additional editing vectors, such as editing vectors X, Y, and Z.That is that double-edited cells AB may be combined with and edited byvectors X, Y, and Z to produce triple-edited edited cells ABX, ABY, andABZ; double-edited cells AC may be combined with and edited by vectorsX, Y, and Z to produce triple-edited cells ACX, ACY, and ACZ; anddouble-edited cells AD may be combined with and edited by vectors X, Y,and Z to produce triple-edited cells ADX, ADY, and ADZ, and so on. Inthis process, many permutations and combinations of edits can beexecuted, leading to very diverse cell populations and cell libraries.In any recursive process, it is advantageous to “cure” the previousengine and editing vectors (or single engine+editing vector in a singlevector system). “Curing” is a process in which one or more vectors usedin the prior round of editing is eliminated from the transformed cells.Curing can be accomplished by, e.g., cleaving the vector(s) using acuring plasmid thereby rendering the editing and/or engine vector (orsingle, combined vector) nonfunctional; diluting the vector(s) in thecell population via cell growth (that is, the more growth cycles thecells go through, the fewer daughter cells will retain the editing orengine vector(s)), or by, e.g., utilizing a heat-sensitive origin ofreplication on the editing or engine vector (or combined engine+editingvector). The conditions for curing will depend on the mechanism used forcuring; that is, in this example, how the curing plasmid cleaves theediting and/or engine plasmid.

FIG. 11 is a simplified block diagram of an embodiment of an exemplaryautomated multi-module cell processing instrument 1100 comprising, e.g.,a bulk liquid growth module for induced editing and enrichment foredited cells. (See, e.g., U.S. Ser. No. 16/545,097, filed 20 Aug. 2019.)The cell processing instrument 1100 may include a housing 1144, areservoir of cells to be transformed or transfected 1102, and a growthmodule (a cell growth device) 1104. The cells to be transformed aretransferred from a reservoir 1102 to the growth module 1104 to becultured until the cells hit a target OD. Once the cells hit the targetOD, the growth module may cool or freeze the cells for later processing,or the cells may be transferred to a cell concentration module 1130where the cells are rendered electrocompetent and concentrated to avolume optimal for cell transformation. Once concentrated, the cells arethen transferred to an electroporation module 1108 (e.g.,transformation/transfection module).

In addition to the reservoir 1102 for storing the cells, the instrument1100 may include a reservoir for storing editing cassettes 1116 and areservoir for storing an expression vector backbone 1118. Both theediting oligonucleotide cassettes and the expression vector backbone aretransferred from the reagent cartridge to a nucleic acid assembly module1120, where the editing oligonucleotide cassettes are inserted into theexpression vector backbone. The assembled nucleic acids may betransferred into an optional purification module 1122 for desaltingand/or other purification and/or concentration procedures needed toprepare the assembled nucleic acids for transformation. Alternatively,pre-assembled nucleic acids, e.g., an editing vector, may be storedwithin reservoir 1116 or 1118. Once the processes carried out by thepurification module 1122 are complete, the assembled nucleic acids aretransferred to, e.g., an electroporation device or module 1108, whichalready contains the cell culture grown to a target OD and renderedelectrocompetent via cell concentration module 1130. In electroporationdevice 1108, the assembled nucleic acids are introduced into the cells.Following electroporation, the cells are transferred into a combinedrecovery/selection module 1110.

Following recovery, and, optionally, selection, the cells aretransferred to a growth, induction, and editing module (bulk liquidculture) 1140. The cells are allowed to grow until the cells reach thestationary growth phase (or nearly so), then editing is induced byinduction of transcription of one or both of the nuclease and gRNA. Insome embodiments, editing is induced by transcription of one or both ofthe nuclease and the gRNA being under the control of an induciblepromoter. In some embodiments, the inducible promoter is a pL promoterwhere the promoter is activated by a rise in temperature and“deactivated” by lowering the temperature.

The recovery, selection, growth, induction, editing and storage modulesmay all be separate, may be arranged and combined as shown in FIG. 11,or may be arranged or combined in other configurations. In certainembodiments, recovery and selection are performed in one module, andgrowth, editing, and re-growth are performed in a separate module.Alternatively, recovery, selection, growth, editing, and re-growth areperformed in a single module.

Once the cells are edited and re-grown (e.g., recovered from editing),the cells may be stored, e.g., in a storage module 1112, where the cellscan be kept at, e.g., 4° C. until the cells are retrieved for furtherstudy (e.g., cell retrieval 1114). Alternatively, the cells may be usedin another round of editing. The multi-module cell processing instrument1100 is controlled by a processor 1142 configured to operate theinstrument based on user input, as directed by one or more scripts, oras a combination of user input or a script. The processor 1142 maycontrol the timing, duration, temperature, and operations of the variousmodules of the instrument 1100 and the dispensing of reagents. Forexample, the processor 1142 may cool the cells post-transformation untilediting is desired, upon which time the temperature may be raised to atemperature conducive of genome editing and cell growth. The processormay be programmed with standard protocol parameters from which a usermay select, a user may specify one or more parameters manually, or oneor more scripts associated with the reagent cartridge may specify one ormore operations and/or reaction parameters. In addition, the processormay notify the user (e.g., via an application to a smart phone or otherdevice) that the cells have reached the target OD, as well as update theuser as to the progress of the cells in the various modules in themulti-module system.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention, nor are theyintended to represent or imply that the experiments below are all of orthe only experiments performed. It will be appreciated by personsskilled in the art that numerous variations and/or modifications may bemade to the invention as shown in the specific aspects without departingfrom the spirit or scope of the invention as broadly described. Thepresent aspects are, therefore, to be considered in all respects asillustrative and not restrictive.

Example I: Growth in the Cell Growth Module

One embodiment of the cell growth device as described herein was testedagainst a conventional cell shaker shaking a 5 ml tube and an orbitalshaker shaking a 125 ml baffled flask to evaluate cell growth inbacterial and yeast cells. Additionally, growth of a bacterial cellculture and a yeast cell culture was monitored in real time using anembodiment of the cell growth device described herein.

In a first example, 20 ml EC23 cells (E. coli cells) in LB were grown ina 35 ml rotating growth vial at 30° C. using the cell growth device asdescribed herein. The rotating growth vial was spun at 600 rpm andoscillated (i.e., the rotation direction was changed) every 1 second. ODwas measured in the cell growth device. In parallel, 5 ml EC23 cells inLB were grown in an orbital shaker in a 5 ml tube at 30° C. and wereshaken at 750 rpm; the OD₆₀₀ was measured at intervals using a NanoDrop™spectrophotometer (Thermo Fisher Scientific). The rotating growthvial/cell growth device performed better than the cell shaker in growingthe cells to OD₆₀₀ 2.6 in slightly over 4 hours (data not shown).

Two additional experiments were performed, this time comparing therotating growth vial/cell growth device with paddles to a baffled flaskand an orbital shaker. In one experiment, 20 ml EC138 cells (E. colicells) in LB were grown in a 35 ml rotating growth vial with a 4-paddleconfiguration at 30° C. The rotating growth vial was spun at 600 rpm andoscillated (i.e., the rotation direction was changed) every 1 second. Inparallel, 20 ml EC138 cells in LB were grown in a 125 ml baffled flaskat 30° C. using an orbital shaker. OD₆₀₀ was measured at intervals usinga NanoDrop™ spetrophotometer (Thermo Fisher Scientific). The resultsdemonstrated that the rotating growth vial/cell growth device performedas well as the orbital shaker in growing the cells to OD₆₀₀ 1.0. In asecond experiment, 20 ml EC138 cells (E. coli cells) in LB were grown ina 35 ml rotating growth vial with a 2-paddle configuration at 30° C.using the cell growth device as described herein. The rotating growthvial was spun at 600 rpm and oscillated (i.e., the rotation directionwas changed) every 1 second. In parallel, 20 ml EC138 cells in LB weregrown in a 125 ml baffled flask at 30° C. using an orbital shaker. OD₆₀₀was measured at intervals using a NanoDrop™ spectrophotometer (ThermoFisher Scientific). The results demonstrated that the rotating growthvial/cell growth device performed as well—or better—as the orbitalshaker in growing the cells to OD₆₀₀ 1.2.

In yet another experiment, the rotating growth vial/cell growth devicewas used to measure OD₆₀₀ in real time. The results of real timemeasurement of growth of an EC138 cell culture at 30° C. usingoscillating rotation and employing a 2-paddle rotating growth vial werethat OD₆₀₀ 2.6 was reached in 4.4 hours.

In another experiment, the rotating growth vial/cell growth device wasused to measure OD₆₀₀ in real time of yeast s288c cells in YPAD. Thecells were grown at 30° C. using oscillating rotation and employing a2-paddle rotating growth vial. For the yeast cells, OD₆₀₀ 6.0 wasreached in 14 hours.

Example II: Cell Concentration

The TFF module as described above in relation to FIGS. 6B-6F has beenused successfully to process and perform buffer exchange on both E. coliand yeast cultures. In concentrating an E. coli culture, the followingsteps were performed:

First, a 20 ml culture of E. coli in LB grown to OD 0.5-0.62 was passedthrough the TFF device in one direction, then passed through the TFFdevice in the opposite direction. At this point, the cells wereconcentrated to a volume of approximately 5 ml. Next, 50 ml of 10%glycerol was added to the concentrated cells, and the cells were passedthrough the TFF device in one direction, in the opposite direction, andback in the first direction for a total of three passes. Again the cellswere concentrated to a volume of approximately 5 ml. Again, 50 ml of 10%glycerol was added to the 5 ml of cells and the cells were passedthrough the TFF device for three passes. This process was repeated; thatis, again 50 ml 10% glycerol was added to cells concentrated to 5 ml,and the cells were passed three times through the TFF device. At the endof the third pass of the three 50 ml 10% glycerol washes, the cells wereagain concentrated to approximately 5 ml of 10% glycerol. The cells werethen passed in alternating directions through the TFF device three moretimes, wherein the cells were concentrated into a volume ofapproximately 400 μl.

Filtrate conductivity and filter processing time was measured for E.coli. Filter performance was quantified by measuring the time and numberof filter passes required to obtain a target solution electricalconductivity. Cell retention was determined by comparing the opticaldensity (OD600) of the cell culture both before and after filtration.Filter health was monitored by measuring the transmembrane flow rateduring each filter pass. Target conductivity (˜16 S/cm) was achieved inapproximately 30 minutes utilizing three 50 ml 10% glycerol washes andthree passes of the cells through the device for each wash. The volumeof the cells was reduced from 20 ml to 400 μl, and recovery ofapproximately 90% of the cells has been achieved.

The same process was repeated with yeast cell cultures. A yeast culturewas initially concentrated to approximately 5 ml using two passesthrough the TFF device in opposite directions. The cells were washedwith 50 ml of 1M sorbitol three times, with three passes through the TFFdevice after each wash. After the third pass of the cells following thelast wash with 1M sorbitol, the cells were passed through the TFF devicetwo times, wherein the yeast cell culture was concentrated toapproximately 525 μl. The filter buffer exchange performance for yeastcells was determined by measuring filtrate conductivity and filterprocessing time. Target conductivity (˜10 S/cm) was achieved inapproximately 23 minutes utilizing three 50 ml 1M sorbitol washes andthree passes through the TFF device for each wash. The volume of thecells was reduced from 20 ml to 525 μl. Recovery of approximately 90% ofthe cells has been achieved.

Example III: Production and Transformation of Electrocompetent E. coliand S. cerevisiae

For testing transformation in the FTEP device, electrocompetent E. colicells were created. To create a starter culture, 6 ml volumes of LBchlor-25 (LB with 25 μg/ml chloramphenicol) were transferred to 14 mlculture tubes. A 25 μl aliquot of E. coli was used to inoculate the LBchlor-25 tubes. Following inoculation, the tubes were placed at a 450angle in the shaking incubator set to 250 RPM and 30° C. for overnightgrowth, between 12-16 hrs. The OD600 value should be between 2.0 and4.0. A 1:100 inoculum volume of the 250 ml LB chlor-25 tubes weretransferred to four sterile 500 ml baffled shake flasks, i.e., 2.5 mlper 250 ml volume shake flask. The flasks were placed in a shakingincubator set to 250 RPM and 30° C. The growth was monitored bymeasuring OD600 every 1 to 2 hr. When the OD600 of the culture wasbetween 0.5-0.6 (approx. 3-4 hrs), the flasks were removed from theincubator. The cells were centrifuged at 4300 RPM, 10 min, 4° C. Thesupernatant was removed, and 100 ml of ice-cold 10% glycerol wastransferred to each sample. The cells were gently resuspended, and thewash procedure performed three times, each time with the cellsresuspended in 10% glycerol. After the fourth centrifugation, the cellresuspension was transferred to a 50 ml conical Falcon tube andadditional ice-cold 10% glycerol added to bring the volume up to 30 ml.The cells were again centrifuged at 4300 RPM, 10 min, 4° C., thesupernatant removed, and the cell pellet resuspended in 10 ml ice-coldglycerol. The cells are aliquoted in 1:100 dilutions of cell suspensionand ice-cold glycerol.

For further testing transformation of the FTEP device, S. Cerevisiaecells were created using the methods as generally set forth inBergkessel and Guthrie, Methods Enzymol., 529:311-20 (2013). Briefly,YPD or YPAD media was inoculated for overnight growth from colonies on aYP+glycerol agar plate to produce 150 mL of cells. The followingmorning, the overnight culture was diluted to an OD600 of approximately0.3. Cells were incubated at 30° C. in a shaking incubator until theyreached an OD600 of 1.5+/−0.1.

A conditioning buffer was prepared using 100 mM lithium acetate and 10mM dithiothreitol. A total of 100 mL of buffer were prepared for every100 mL of cells grown. Cells were harvested in 250 mL bottles bycentrifugation at 4300 rpm for 6 minutes, and the supernatant removed.The cells were suspended in conditioning buffer, then the suspensiontransferred into an appropriate flask and shaken at 200 RPM and 30° C.for 30 minutes. The suspension was transferred to 250 mL bottles andspun at 4300 rpm for 6 minutes. The supernatant was removed and thepellets resuspended in cold 1 M sorbitol. These steps were repeatedthree times for a total of three wash-spin-decant steps. The pellet wassuspended in sorbitol so that 20 mL of OD 1.5 culture were resuspendedin 500 μL. For each 500 μL volume of resuspended cells, a 100 μL volumecontaining DNA and Tween80 was added to the cell suspension.

A comparative electroporation experiment was performed to determine theefficiency of transformation of electrocompetent S. cerevisiae and E.coli using the obstruction array FTEP device described, benchmarkedagainst a NEPA electroporation device and a single constriction FTEP.See FIG. 12A for a comparison of the single constriction FTEP device andthe obstruction array FTEP device. In the obstruction array FTEP devicetested, ramps in the central region (one before and one after the arraymoving from the inlet proximal region of the flow channel toward theoutlet proximal region of the flow channel) decreases the channel heightfrom 100 μm near the electrode channels to 50 μm at the obstruction. Theflow rate was controlled with a pressure control system, and theperformance of the obstruction array FTEP device was tested at variousvoltages and pressures. The suspension of cells with DNA was loaded intothe FTEP inlet reservoir. The transformed cells flowed directly from theinlet and inlet channel, through the flow channel, through the outletchannel, and into the outlet. The cells were transferred into a tubecontaining additional recovery medium and placed in an incubator shakerat 30° C. shaking at 250 rpm for 3 hours. The cells were plated todetermine the colony forming units (CFUs) that survived electroporationand the CFUs that survived electroporation and took up a plasmid. Plateswere incubated at 30° C.; E. coli colonies were counted afterapproximately 24 hrs.

FIG. 12B shows data revealing that the obstruction array FTEP devicedemonstrated equivalent uptake of DNA by S. cerevisiae as compared tothe single constriction FTEP device and a NEPA (cuvette) device. Thesingle left-most bar indicates the cell input for each datapoint. Foreach datapoint, the left bar indicates the number of cells that survivedelectroporation, and the right bar indicates the number of cells thatwere transformed with the DNA. Replicates were performed for eachdatapoint and the bars are the means of the duplicates. FIG. 12C showsdata revealing that a different obstruction array FTEP device with aminimum flow path width of 100 μm and minimum height of 50 μmdemonstrated uptake of DNA by S. cerevisiae within a factor of 1.3 ascompared to the single constriction FTEP device. The single left-mostbar indicates the cell input for each datapoint. For each datapoint, theleft bar indicates the number of cells that survived electroporation,and the right bar indicates the number of cells that were transformedwith the DNA. Replicates were performed for each datapoint and the barsare the means of the duplicates. FIG. 12D shows data revealing that anobstruction array FTEP device with a minimum flow path width of 40 μmand a minimum height of 50 μm demonstrated uptake of DNA by E. coliwithin a factor of 5 as compared to the single constriction FTEP deviceand a NEPA (cuvette) device. The single left-most bar indicates the cellinput for each datapoint. For each datapoint, the left bar indicates thenumber of cells that survived electroporation (bars with angled linesfrom bottom left to top right), and the right bar indicates the numberof cells that were transformed with the editing plasmid (i.e., uptake)(bars with angled lines from top left to bottom right). Replicates wereperformed for each datapoint and the bars are the means of theduplicates.

Example IV: Optimization of FTEP Configuration

For optimizing transformation in the parallel-obstruction FTEP deviceembodiment shown in FIG. 1L-1N, electrocompetent S288c cells werecreated. FIG. 13 shows at left a plot of simulated electric fieldstrength vs. residence time for the “hour glass”-shaped FTEP embodimentpictured above the plot (conducted at 2.75 kV, 3 psi). The plot showsthe energy electric field strength (kV/cm) experienced by cells at d=0(at the center of the FTEP device), d=12.5 μm (from the center), d=20 μm(from the center), and d=22.5 μm (from the center). The dotted line atapproximately 9 kV/cm shows the electric field strength experienced bycells porated in a Nepa Gene cuvette. Note that in the “hour glass” FTEPconfiguration, the cells experience a spike in electric field strengthsignificantly above that experienced in a cuvette. FIG. 13 at rightshows a plot of simulated electric field strength vs. residence time forthe “abrupt step” FTEP embodiment shown above the plot (conducted at 2.0kV, 0.9 psi). Note that with this embodiment of an FTEP, it is possibleto control the magnitude of a constant electric field strengthexperienced by the cells. Again, the plot shows the electric fieldstrength E (kV/cm) experienced by cells at d=0 (at the center of theFTEP device), d=12.5 μm (from the center), d=20 μm (from the center),and d=22.5 μm (from the center). The dotted line at approximately 9kV/cm shows the electric field strength experienced by cells porated ina NepaGene cuvette.

FIG. 14 shows the results of a sweep of various electric field strengthsand residence times with S288c, achieved by varying the applied voltageand pressure, for the parallel-obstruction FTEP embodiment shown in FIG.1L-1N. The right plot of FIG. 13 demonstrates that the “abrupt step”configuration of FTEP allows for control over the magnitude of aconstant electric field strength experienced by cells in the FTEPdevice; however, though the “abrupt step” configuration allows forcontrol of field strength, the configuration of a single “abrupt step”can lead to clogging of the flow channel, which in turn leads tocatastrophic failure of the FTEP device. To decrease the likelihood ofclogging while still maintaining a channel configuration that allowstuning of field strength and residence time, the parallel-obstructionFTEP embodiment shown in FIG. 1L-1N was tested. In this embodiment, theobstructions are elongated ovals (1.0 mm in length) which form 8parallel “lanes” in the center region of the flow channel. Note that forthe 1.0 mm-long center obstruction configuration the optimal appliedvoltage was 3 kV with an applied pressure of 6.8 psi, leading to anuptake value of 8.51E+04 CFUs.

FIG. 15 shows at top the results of a sweep of various electric fieldstrengths and residence times with S288c, achieved by varying theapplied voltage and pressure, for the parallel-obstruction FTEPembodiment shown in FIG. 1L-1N. In this embodiment, the obstructions areelongated ovals that are 0.5 mm in length, which form 8 parallel “lanes”in the center region of the flow channel. Note that the optimal appliedvoltage was 1.5 kV with an applied pressure of 2.6 psi, leading to anuptake value of 2.55E+04 CFUs. At top shows a comparison between theparallel-obstruction FTEP embodiment shown in FIG. 1L-1N with 1.0mm-long obstructions vs. 0.5 mm-long obstructions. Note that themagnitude of a constant electric field strength and residence timewithin the electric field can be controlled in this embodiment FTEP byadjusting the applied voltage and pressure.

Example V: Fully-Automated Singleplex RGN-Directed Editing Run

Singleplex automated genomic editing using MAD7 nuclease wassuccessfully performed with an automated multi-module instrument of thedisclosure. See U.S. Pat. No. 9,982,279; and U.S. Ser. No. 16/024,831filed 30 Jun. 2018; Ser. No. 16/024,816 filed 30 Jun. 2018; Ser. No.16/147,353 filed 28 Sep. 2018; Ser. No. 16/147,865 filed 30 Sep. 2018;and Ser. No. 16/147,871 filed 30 Jun. 2018.

An ampR plasmid backbone and a lacZ_F172* editing cassette wereassembled via Gibson Assembly® into an “editing vector” in an isothermalnucleic acid assembly module included in the automated instrument.lacZ_F172 functionally knocks out the lacZ gene. “lacZ_F172*” indicatesthat the edit happens at the 172nd residue in the lacZ amino acidsequence. Following assembly, the product was de-salted in theisothermal nucleic acid assembly module using AMPure beads, washed with80% ethanol, and eluted in buffer. The assembled editing vector andrecombineering-ready, electrocompetent E. Coli cells were transferredinto a transformation module for electroporation. The cells and nucleicacids were combined and allowed to mix for 1 minute, and electroporationwas performed for 30 seconds. The parameters for the poring pulse were:voltage, 2400 V; length, 5 ms; interval, 50 ms; number of pulses, 1;polarity, +. The parameters for the transfer pulses were: Voltage, 150V; length, 50 ms; interval, 50 ms; number of pulses, 20; polarity, +/−.Following electroporation, the cells were transferred to a recoverymodule (another growth module) and allowed to recover in SOC mediumcontaining chloramphenicol. Carbenicillin was added to the medium after1 hour, and the cells were allowed to recover for another 2 hours. Afterrecovery, the cells were held at 4° C. until recovered by the user.

After the automated process and recovery, an aliquot of cells was platedon MacConkey agar base supplemented with lactose (as the sugarsubstrate), chloramphenicol and carbenicillin and grown until coloniesappeared. White colonies represented functionally edited cells, purplecolonies represented un-edited cells. All liquid transfers wereperformed by the automated liquid handling device of the automatedmulti-module cell processing instrument.

The result of the automated processing was that approximately 1.0E⁻⁰³total cells were transformed (comparable to conventional benchtopresults), and the editing efficiency was 83.5%. The lacZ_172 edit in thewhite colonies was confirmed by sequencing of the edited region of thegenome of the cells. Further, steps of the automated cell processingwere observed remotely by webcam and text messages were sent to updatethe status of the automated processing procedure.

Example VI: Fully-Automated Recursive Editing Run

Recursive editing was successfully achieved using the automatedmulti-module cell processing instrument. An ampR plasmid backbone and alacZ_V10* editing cassette were assembled via Gibson Assembly® into an“editing vector” in an isothermal nucleic acid assembly module includedin the automated system. Similar to the lacZ_F172 edit, the lacZ_V10edit functionally knocks out the lacZ gene. “lacZ_V10” indicates thatthe edit happens at amino acid position 10 in the lacZ amino acidsequence. Following assembly, the product was de-salted in theisothermal nucleic acid assembly module using AMPure beads, washed with80% ethanol, and eluted in buffer. The first assembled editing vectorand the recombineering-ready electrocompetent E. coli cells weretransferred into a transformation module for electroporation. The cellsand nucleic acids were combined and allowed to mix for 1 minute, andelectroporation was performed for 30 seconds. The parameters for theporing pulse were: voltage, 2400 V; length, 5 ms; interval, 50 ms;number of pulses, 1; polarity, +. The parameters for the transfer pulseswere: Voltage, 150 V; length, 50 ms; interval, 50 ms; number of pulses,20; polarity, +/−. Following electroporation, the cells were transferredto a recovery module (another growth module) allowed to recover in SOCmedium containing chloramphenicol. Carbenicillin was added to the mediumafter 1 hour, and the cells were grown for another 2 hours. The cellswere then transferred to a centrifuge module and a media exchange wasthen performed. Cells were resuspended in TB containing chloramphenicoland carbenicillin where the cells were grown to OD600 of 2.7, thenconcentrated and rendered electrocompetent.

During cell growth, a second editing vector was prepared in theisothermal nucleic acid assembly module. The second editing vectorcomprised a kanamycin resistance gene, and the editing cassettecomprised a galK Y145* edit. If successful, the galK Y145* edit conferson the cells the ability to uptake and metabolize galactose. The editgenerated by the galK Y154* cassette introduces a stop codon at the154th amino acid reside, changing the tyrosine amino acid to a stopcodon. This edit makes the galK gene product non-functional and inhibitsthe cells from being able to metabolize galactose. Following assembly,the second editing vector product was de-salted in the isothermalnucleic acid assembly module using AMPure beads, washed with 80%ethanol, and eluted in buffer. The assembled second editing vector andthe electrocompetent E. Coli cells (that were transformed with andselected for the first editing vector) were transferred into atransformation module for electroporation, using the same parameters asdetailed above. Following electroporation, the cells were transferred toa recovery module (another growth module), allowed to recover in SOCmedium containing carbenicillin. After recovery, the cells were held at4° C. until retrieved, after which an aliquot of cells were plated on LBagar supplemented with chloramphenicol, and kanamycin. To quantify bothlacZ and galK edits, replica patch plates were generated on two mediatypes: 1) MacConkey agar base supplemented with lactose (as the sugarsubstrate), chloramphenicol, and kanamycin, and 2) MacConkey agar basesupplemented with galactose (as the sugar substrate), chloramphenicol,and kanamycin. All liquid transfers were performed by the automatedliquid handling device of the automated multi-module cell processinginstrument.

In this recursive editing experiment, 41% of the colonies screened hadboth the lacZ and galK edits, the results of which were comparable tothe double editing efficiencies obtained using a “benchtop” or manualapproach.

While this invention is satisfied by embodiments in many differentforms, as described in detail in connection with preferred embodimentsof the invention, it is understood that the present disclosure is to beconsidered as exemplary of the principles of the invention and is notintended to limit the invention to the specific embodiments illustratedand described herein. Numerous variations may be made by persons skilledin the art without departure from the spirit of the invention. The scopeof the invention will be measured by the appended claims and theirequivalents. The abstract and the title are not to be construed aslimiting the scope of the present invention, as their purpose is toenable the appropriate authorities, as well as the general public, toquickly determine the general nature of the invention. In the claimsthat follow, unless the term “means” is used, none of the features orelements recited therein should be construed as means-plus-functionlimitations pursuant to 35 U.S.C. § 112, ¶6.

We claim:
 1. A flow-through electroporation (FTEP) device forintroducing an exogenous material into cells in a fluid, the FTEP devicecomprising: a. an inlet and an inlet channel for receiving a fluidcomprising cells and/or exogenous material into the FTEP device; b. anoutlet and an outlet channel for removing a fluid comprising transformedcells and exogenous material from the FTEP device; c. a flow channelintersecting and positioned between the inlet channel and the outletchannel, wherein the flow channel has, moving from the inlet channeltoward the outlet channel, an inlet-filter region, an inlet-proximalregion, a central region, an outlet-proximal region, and anoutlet-filter region; d. an inlet filter comprising filter elementsdisposed in the inlet-filter region of the flow channel and an outletfilter comprising filter elements disposed in the outlet-filter regionof the flow channel; e. a plurality of parallel-configured obstructionsdefining flow lanes disposed within the central region of the flowchannel; and f. a first and a second electrode positioned in electrodechannels, wherein the first electrode is positioned in the inletproximal region of the flow channel and the second electrode ispositioned in the outlet proximal region of the flow channel; whereinthe electrodes are positioned perpendicularly to the flow channel, arein fluid and electrical communication with fluid in the flow channel,wherein the electrodes apply one or more electric pulses to the cells inthe fluid as they pass through the flow channel, thereby introducingexogenous material into the cells in the fluid, and wherein the deviceis formed by injection molding in one piece with the exception of theelectrodes.
 2. The FTEP device of claim 1, wherein there are three ormore parallel-configured obstructions.
 3. The FTEP device of claim 1,wherein the parallel-configured obstructions are elongated oval-shaped.4. The FTEP device of claim 3, wherein the FTEP device comprises asecond inlet and a second inlet channel and further comprising areservoir coupled to the second inlet for introducing exogenous materialinto the FTEP device.
 5. The FTEP device of claim 4, wherein the secondinlet and second inlet channel are located between the inlet channel andthe first electrode.
 6. The FTEP device of claim 4, wherein the secondinlet and second inlet channel are located between the second electrodeand the outlet channel.
 7. The FTEP device of claim 1, furthercomprising a reservoir coupled to the inlet for introducing the cells influid into the FTEP device and a reservoir coupled to the outlet forremoving transformed cells from the FTEP device.
 8. The FTEP device ofclaim 1, wherein device is configured for use with bacterial, yeast andmammalian cells.
 9. The FTEP device of claim 1, wherein the number ofobstructions in the central region of the flow channel is from 3 to 15.10. The FTEP device of claim 9, wherein the narrowest flow path betweenobstructions is from 30 to 250 μm wide.
 11. The FTEP device of claim 1,wherein the narrowest flow path between obstructions is from 10 to 350μm wide.
 12. The FTEP device of claim 1, wherein the electrodes supply avoltage of 1-60 kV/cm.
 13. The FTEP device of claim 12, wherein theelectrodes supply a voltage of 5-40 kV/cm.
 14. The FTEP device of claim1, wherein the flow through the FTEP device is from 0.01 mL/min to 7.5mL/min.
 15. The FTEP device of claim 1, wherein the pressure in the FTEPis from 1-30 psi.
 16. The FTEP device of claim 15, wherein the pressurein the FTEP is from 2-10 psi.
 17. The FTEP device of claim 1, whereinthe FTEP is from 3-15 cm long.
 18. The FTEP device of claim 1, whereinthe FTEP is from 0.5 to 5 cm wide.
 19. The FTEP device of claim 1,further comprising a ramp in the central region proximal to theinlet-proximal region of the flow channel to a central portion of thecentral region decreasing the flow channel height, and a ramp from thecentral portion of the central region to the central region proximal tothe outlet-proximal region of the flow channel increasing the flowchannel height.